JP2023508942A - Proteins that bind NKG2D, CD16 and CLEC12A - Google Patents

Abstract

Described herein are multispecific binding proteins that bind to the NKG2D receptor, CD16, and CLEC12A, pharmaceutical compositions comprising the multispecific binding proteins, and therapeutic methods useful in the treatment of cancer. be. The present application provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and CLEC12A. Such proteins can engage more than one NK-activated receptor and block the binding of natural ligands to NKG2D.

Description

This application claims priority to US Provisional Application No. 63/020,798, filed May 6, 2020, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING The Sequence Listing in computer readable format (CRF) in ASCII text format is incorporated herein by reference in its entirety. The sequence listing text file is titled "14247-540-228_SEQ_LISTING", was created May 3, 2021 and is 291,375 bytes in size.

FIELD OF THE INVENTION This application relates to multispecific binding proteins that bind to NKG2D, CD16 and CLEC12A on cells, pharmaceutical compositions comprising such proteins and such proteins and pharmaceutical compositions, including the treatment of cancer. It relates to a method of treatment using

Despite considerable research efforts, cancer continues to represent a significant clinical and financial burden in countries around the world. According to the World Health Organization (WHO), it is the second leading cause of death. Surgery, radiotherapy, chemotherapy, biologic therapy, immunotherapy, hormone therapy, stem cell transplantation, and precision medicine are among the existing treatments. Despite extensive research in these areas, highly effective therapeutic solutions, especially for the most aggressive cancers, have yet to be identified. In addition, many existing anti-cancer therapies have significant adverse side effects.

Cancer immunotherapy is desirable because it is highly specific and can use the patient's own immune system to promote the destruction of cancer cells. Fusion proteins, such as bispecific T cell engagers, are cancer immunotherapies described in the literature that bind to tumor cells and T cells and promote their destruction. Antibodies that bind to specific tumor-associated antigens have been described in the literature. See for example WO2016/134371 and WO2015/095412.

Natural killer (NK) cells are components of the innate immune system and account for approximately 15% of circulating lymphocytes. NK cells were originally characterized as having the ability to infiltrate virtually all tissues and effectively kill tumor cells without the need for prior sensitization. Activated NK cells kill target cells in a manner similar to cytotoxic T cells, ie, via cytolytic granules, including perforin and granzymes, and the death receptor pathway. Activated NK cells also secrete inflammatory cytokines such as IFN-γ and chemokines that promote the recruitment of other leukocytes to target tissues.

NK cells respond to signals through various activating and inhibitory receptors on their surface. For example, when NK cells encounter healthy autologous cells, their activity is inhibited by activation of killer cell immunoglobulin-like receptors (KIRs). Alternatively, when NK cells encounter foreign or cancer cells, they are activated through their activating receptors (eg, NKG2D, NCR, DNAM1). NK cells are also activated by the constant regions of some immunoglobulins through the CD16 receptor on their surface. The overall susceptibility to NK cell activation depends on the summation of stimulatory and inhibitory signals. NKG2D is a type II transmembrane protein expressed by essentially all natural killer cells, and NKG2D functions as an activating receptor. NKG2D is also found on T cells that function as co-stimulatory receptors. The ability to modulate NK cell function through NKG2D is useful in a variety of therapeutic settings, including malignancies.

C-type lectin domain family 12 member A (CLEC12A), also known as C-type lectin-like molecule-1 (CLL-1) or myeloid inhibitory C-type lectin-like receptor (MICL), is a C-type lectin/C It is a member of the type lectin-like domain (CTL/CTLD) superfamily. Members of this family share a common protein fold and have diverse functions such as cell adhesion, cell-cell signaling, glycoprotein turnover, and roles in inflammation and immune responses. CLEC12A, a type II transmembrane glycoprotein, is overexpressed in more than 90% of acute myeloid leukemia patients in leukemic stem cells, but not in normal hematopoietic cells.
Despite numerous efforts made by several biotechnology and pharmaceutical companies, the development of specific CLEC12A-targeted biologics has been hampered by the absence of antibodies with good developability properties. there is A challenge in discovering CLEC12A antibodies may be due to the complexity of the antigen. CLEC12A is a monomeric, highly glycosylated protein with six potential N-glycosylation sites within the 201 amino acid extracellular domain. Four of the six N-glycosylation sites are clustered in the membrane-proximal domain of the molecule and may be involved in target presentation on the cell surface. Variation in the glycosylation status of CLEC12A on the surface of different cell types has been reported (Marshall et al., (2006) Eur J Immunol. 36(8):2159-69).
Therefore, there remains a need in the field for new and useful proteins that bind to CLEC12A for use in cancer therapy.

WO2016/134371 WO2015/095412

Marshall et al. , (2006) Eur J Immunol. 36(8):2159-69

The present application provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and CLEC12A. Such proteins can engage more than one NK-activated receptor and block the binding of natural ligands to NKG2D. In certain embodiments, the protein is capable of stimulating human NK cells. In some embodiments, the protein can stimulate NK cells in humans and other species such as rodents and cynomolgus monkeys. Also provided are formulations comprising any one of the proteins disclosed herein; cells comprising one or more nucleic acids expressing the protein, and methods of using the protein to enhance tumor cell death.

Accordingly, in one aspect, the application provides proteins comprising:
(a) a first antigen binding site that binds to NKG2D;
(b) a second antigen binding site that binds CLEC12A; and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen binding site that binds CD16;
Here, the second antigen binding site that binds to CLEC12A includes:
(i) a heavy chain comprising complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3), each comprising the amino acid sequences of SEQ ID NOS: 137, 272, and 273; a variable domain (VH); and a light chain variable domain (VL) comprising CDR1, CDR2 and CDR3, each comprising the amino acid sequences of SEQ ID NOs: 140, 141 and 142;
(ii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139 respectively; VL containing;
(iii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOS: 251, 196 and 246 respectively; VL containing;
(v) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:221, 166 and 223 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:240, 226 and 179 respectively VL containing;
(v) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:206, 166 and 241 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:245, 239 and 179 respectively VL containing;
(vi) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:221, 236 and 223 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:152, 226 and 179 respectively; VL containing;
(vii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 159, 170 and 183 respectively; VL containing;
(viii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 197, 202 and 207 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 211, 212 and 214 respectively VL;
(ix) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:220, 222 and 257 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:224, 225 and 227 respectively; or (x) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOS:230, 231 and 232 respectively; and CDR1 comprising the amino acid sequences of SEQ ID NOS:233, 234 and 235 respectively , CDR2, and CDR3.

In some embodiments, the second antigen binding site that binds CLEC12A is a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 272, and 273, respectively; VL comprising CDR1, CDR2, and CDR3, comprising 141 and 142 amino acid sequences. In some embodiments, the second antigen binding site that binds CLEC12A is a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138, and 139, respectively; and SEQ ID NO: 140, respectively; VL comprising CDR1, CDR2 and CDR3 comprising 141 and 142 amino acid sequences.

In certain embodiments, VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:335 and VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:336. In certain embodiments, VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:270 and VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:271. In certain embodiments, VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:178 and VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:289. In certain embodiments, VH comprises the amino acid sequence of SEQ ID NO:335 and VL comprises the amino acid sequence of SEQ ID NO:336. In certain embodiments, VH comprises the amino acid sequence of SEQ ID NO:270 and VL comprises the amino acid sequence of SEQ ID NO:271. In certain embodiments, VH comprises the amino acid sequence of SEQ ID NO:178 and VL comprises the amino acid sequence of SEQ ID NO:289. 147 and 144; 244 and 144; 178 and 144; 256 and 144; 143 and 163; 143 and 167; each containing an amino acid sequence.

In some embodiments, the second antigen binding site is present as a single chain fragment variable (scFv), wherein the scFv comprises 156, 157, 160, 161, 164, 165, 168, 169, 172, 173, 176, 177, 180, and 181. In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:274. In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:180.

In some embodiments, the second antigen binding site that binds CLEC12A is a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 251, 196, and 246, respectively; VL comprising CDR1, CDR2, and CDR3, comprising 199, and 201 amino acid sequences.

In certain embodiments, the second antigen binding site that binds CLEC12A comprises:
(i) VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 195, 196, 187 respectively; VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 198, 199, 201 respectively; ;
(ii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 192, 196 and 187 respectively; or (iii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 192, 196 and 213 respectively; and CDR1 comprising the amino acid sequences of SEQ ID NOs 198, 199 and 201 respectively VL containing CDR2 and CDR3.

In certain embodiments, VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:148 and VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:203.

In certain embodiments, VH comprises the amino acid sequence of SEQ ID NO:148 and VL comprises the amino acid sequence of SEQ ID NO:203. In certain embodiments, VH and VL comprise the amino acid sequence of SEQ ID NOS: 162 and 203; 210 and 203; 162 and 218; 148 and 218; or 210 and 218, respectively. In some embodiments, the second antigen-binding site is present as an scFv, wherein the scFv comprises It comprises an amino acid sequence selected from 254 and 255. In certain embodiments, the scFv comprises the amino acid sequence of SEQ ID NO:204.

In certain embodiments, the second antigen binding site of the preceding embodiment binds CLEC12A in a glycosylation-independent manner.

In another aspect, the application provides a protein comprising:
(a) a first antigen binding site that binds to NKG2D;
(b) a second antigen binding site that binds CLEC12A; and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen binding site that binds CD16;
Here, a second antigen binding site binds CLEC12A in a manner independent of said glycosylation.

In some embodiments of the proteins disclosed herein, the second antigen binding site binds human CLEC12A with a dissociation constant ( _KD ) of 20 nM or less as measured by surface plasmon resonance (SPR). do. In certain embodiments, the second antigen binding site binds human CLEC12A with a K _D of 1 nM or less, as measured by SPR. In certain embodiments, the second antigen binding site binds human CLEC12A comprising a K244Q substitution.

In some embodiments of the proteins disclosed herein, the protein comprises an antibody Fc domain or portion thereof sufficient to bind CD16.

In some embodiments of the proteins disclosed herein, the first antigen binding site that binds NKG2D is a Fab fragment and the second antigen binding site that binds CLEC12A is a scFv.

In some embodiments of the proteins disclosed herein, the first antigen binding site that binds NKG2D is a scFv and the second antigen binding site that binds CLEC12A is a Fab fragment.

In some embodiments of the proteins disclosed herein, the protein further comprises an additional antigen binding site that binds CLEC12A. In certain embodiments, the first antigen binding site that binds NKG2D is a scFv and the second and additional antigen binding sites that bind CLEC12A are each a Fab fragment. In certain embodiments, the first antigen binding site that binds NKG2D is a scFv and the second and additional antigen binding sites that bind CLEC12A are each scFv. In certain embodiments, the amino acid sequences of the second and additional antigen binding sites are identical. In certain embodiments, the amino acid sequences of the second and additional antigen binding sites are different.

In another aspect, the application provides a protein comprising:
(a) a first antigen binding site that binds to NKG2D;
(b) a second antigen binding site that binds CLEC12A; and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen binding site that binds CD16,
Here, the first antigen binding site that binds NKG2D is the Fab fragment and the second antigen binding site that binds CLEC12A is the scFv.

In some embodiments of the proteins disclosed herein, the scFv that binds NKG2D has sufficient antibody constant domains or partly connected. In certain embodiments, the hinge further comprises the amino acid sequence Thr-Lys-Gly.

In some embodiments of the proteins disclosed herein, each scFv that binds CLEC12A has sufficient antibody constant domains to bind CD16 via a hinge containing Ala-Ser or Gly-Ser. or connected to a part thereof. In certain embodiments, the hinge further comprises the amino acid sequence Thr-Lys-Gly.

In some embodiments of the proteins disclosed herein, the heavy chain variable domain of the scFv forms a disulfide bridge with the light chain variable domain of the scFv within the scFv that binds NKG2D. In certain embodiments, a disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain, numbered under the Kabat numbering scheme.

In some embodiments of the proteins disclosed herein, within each scFv that binds CLEC12A, the scFv heavy chain variable domain forms a disulfide bridge with the scFv light chain variable domain. In certain embodiments, a disulfide bridge is formed between C44 of the heavy chain variable domain and C100 of the light chain variable domain, numbered under the Kabat numbering scheme.

In some embodiments of the proteins disclosed herein, within the scFv that binds NKG2D, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. In certain embodiments, the flexible linker comprises (G ₄ S) ₄ .

In some embodiments of the proteins disclosed herein, within each scFv that binds CLEC12A, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. In certain embodiments, the flexible linker comprises (G ₄ S) ₄ .

In some embodiments of the proteins disclosed herein, the heavy chain variable domain is positioned C-terminal to the light chain variable domain within the scFv that binds NKG2D.

In some embodiments of the proteins disclosed herein, within each scFv that binds CLEC12A, the heavy chain variable domain is positioned C-terminal to the light chain variable domain.

In some embodiments of the proteins disclosed herein, the heavy chain variable domain is positioned N-terminal to the light chain variable domain within the scFv that binds NKG2D.

In some embodiments of the proteins disclosed herein, within each scFv that binds CLEC12A, the heavy chain variable domain is positioned N-terminal to the light chain variable domain.

In some embodiments of the proteins disclosed herein, the Fab fragment that binds NKG2D is not positioned between the antigen binding site and Fc or portion thereof.

In some embodiments of the proteins disclosed herein, the Fab fragment that binds CLEC12A is not located between the antigen binding site and Fc or portion thereof.

In some embodiments of the proteins disclosed herein, the first antigen binding site that binds NKG2D comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:81, 82, and 112, respectively. VH; and VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:86, 77, and 87, respectively.
In certain embodiments, the first antigen binding site that binds NKG2D comprises:
(i) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:81, 82, and 97, respectively; or (ii) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:81, 82, and 84, respectively; and CDR1, comprising the amino acid sequences of SEQ ID NOs:86, 77, and 87, respectively. , CDR2, and CDR3.

In some embodiments of the proteins disclosed herein, the VH of the first antigen binding site comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:95, and VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:85. In certain embodiments, the VH of the first antigen binding site comprises the amino acid sequence of SEQ ID NO:95; and the VL of the first antigen binding site comprises the amino acid sequence of SEQ ID NO:85.

In some embodiments of the proteins disclosed herein, the antibody Fc domain is a human IgG1 antibody Fc domain. In some embodiments, an antibody Fc domain or portion thereof comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:118. In certain embodiments, at least one polypeptide chain of an antibody Fc domain is Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, numbered according to the EU numbering system , K370, N390, K392, T394, D399, S400, D401, F405, Y407, K409, T411 and K439, as compared to SEQ ID NO: 118. . In certain embodiments, at least one polypeptide chain of an antibody Fc domain is Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, numbered according to the EU numbering system, Ｓ３５４Ｃ、Ｅ３５６Ｋ、Ｅ３５７Ｑ、Ｅ３５７Ｌ、Ｅ３５７Ｗ、Ｋ３６０Ｅ、Ｋ３６０Ｗ、Ｑ３６２Ｅ、Ｓ３６４Ｋ、Ｓ３６４Ｅ、Ｓ３６４Ｈ、Ｓ３６４Ｄ、Ｔ３６６Ｖ、Ｔ３６６Ｉ、Ｔ３６６Ｌ、Ｔ３６６Ｍ、Ｔ３６６Ｋ、Ｔ３６６Ｗ、Ｔ３６６Ｓ、Ｌ３６８Ｅ、Ｌ３６８Ａ、Ｌ３６８Ｄ、Ｋ３７０Ｓ、Ｎ３９０Ｄ、Ｎ３９０Ｅ、 Ｋ３９２Ｌ、Ｋ３９２Ｍ、Ｋ３９２Ｖ、Ｋ３９２Ｆ、Ｋ３９２Ｄ、Ｋ３９２Ｅ、Ｔ３９４Ｆ、Ｄ３９９Ｒ、Ｄ３９９Ｋ、Ｄ３９９Ｖ、Ｓ４００Ｋ、Ｓ４００Ｒ、Ｄ４０１Ｋ、Ｆ４０５Ａ、Ｆ４０５Ｔ、Ｙ４０７Ａ、Ｙ４０７Ｉ、Ｙ４０７Ｖ、Ｋ４０９Ｆ、Ｋ４０９Ｗ、Ｋ４０９Ｄ、Ｔ４１１Ｄ、Ｔ４１１Ｅ、Ｋ４３９Ｄ、及びＫ４３９Ｅ relative to SEQ ID NO: 118, comprising one or more mutations selected from In certain embodiments, one polypeptide chain of the antibody heavy chain constant region is Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, K392, T394, D399, S400 , D401, F405, Y407, K409, T411, and K439, relative to SEQ ID NO: 118; other polypeptide chains of the antibody heavy chain constant region are Q347, Y349, L351, S354, E356, E357, S364, T366, L368, K370, N390, K392, T394, D399, D401, F405, Y407, K409, T411, numbered according to the EU numbering system; and K439, as compared to SEQ ID NO: 136. In certain embodiments, one polypeptide chain of the antibody heavy chain constant region comprises the K360E and K409W substitutions relative to SEQ ID NO: 118; contains Q347R, D399V and F405T substitutions compared to SEQ ID NO: 136, numbered according to. In certain embodiments, one polypeptide chain of the antibody heavy chain constant region comprises the Y349C substitution relative to SEQ ID NO: 118; the other polypeptide chain of the antibody heavy chain constant region is numbered according to the EU numbering system. contains the S354C substitution compared to SEQ ID NO: 136 attached.

In another aspect, the application provides proteins comprising:
(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:259;
(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:260; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:261.

In another aspect, the application provides a protein comprising:
(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:276;
(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:260; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:261.

In another aspect, the application provides pharmaceutical compositions comprising a protein disclosed herein and a pharmaceutically acceptable carrier.

In another aspect, the application provides pharmaceutical formulations comprising a protein disclosed herein and a buffer of pH 7.0 or higher. In some embodiments, the pH of the formulation is between 7.0-8.0. In some embodiments, the buffer contains citrate. In certain embodiments, the citrate concentration is 10-20 mM. In some embodiments, the buffer further comprises phosphate. In some embodiments, the buffer further comprises a sugar or sugar alcohol. In certain embodiments, the sugar or sugar alcohol is a disaccharide. In certain embodiments, the disaccharide is sucrose. In some embodiments, the concentration of sugar or sugar alcohol in the pharmaceutical formulation is 200-300 mM. In certain embodiments, the concentration of sugar or sugar alcohol in the pharmaceutical formulation is about 250 mM. In some embodiments, the buffer further comprises a non-ionic surfactant. In certain embodiments, the nonionic surfactant comprises polysorbate. In certain embodiments, the polysorbate is polysorbate 80. In some embodiments, the concentration of polysorbate 80 in the pharmaceutical formulation is 0.005%-0.05%. In certain embodiments, the concentration of polysorbate 80 in the pharmaceutical formulation is about 0.01%. In some embodiments, the concentration of NaCl, if any, is about 1 mM or less in the pharmaceutical formulation. In some embodiments, the concentration of protein is 10-200 mg/mL. In certain embodiments, the protein concentration is about 20 mg/mL.

In some embodiments, the pharmaceutical formulation comprises about 20 mM citrate, about 250 mM sucrose, and about 0.01% polysorbate 80 at pH 7.0. In certain embodiments, the pharmaceutical formulation comprises about 20 mg/mL protein, about 20 mM potassium phosphate, about 10 mM sodium citrate, and about 125 mM sodium chloride at pH 7.8.

In another aspect, the application provides cells comprising one or more nucleic acids encoding the proteins disclosed herein.

In another aspect, the present application provides a method of enhancing tumor cell death, comprising exposing tumor cells and natural killer cells to an effective amount of a protein, pharmaceutical composition, or pharmaceutical formulation disclosed herein I will provide a.

In another aspect, the application provides a method of treating cancer, comprising an effective amount of a protein, pharmaceutical composition, or pharmaceutical formulation thereof disclosed herein Including administering to a subject. In some embodiments, the cancer is a hematologic malignancy. In certain embodiments, the hematologic malignancy is acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), myeloproliferative neoplasm (MPN), lymphoma, Selected from the group consisting of non-Hodgkin's lymphoma and classic Hodgkin's lymphoma.

In some embodiments, AML is undifferentiated acute myeloblastic leukemia, minimally differentiated acute myeloblastic leukemia, differentiated acute myeloblastic leukemia, acute promyelocytic leukemia (APL), acute Myelomonocytic leukemia, acute myelomonocytic leukemia with eosinophilia, acute monocytic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia (AMKL), acute basophilic leukemia, fibrosis Acute panmyelopathy with concomitant and blast plasmacytoid dendritic cell neoplasm (BPDCN). In certain embodiments, AML is characterized by expression of CLL-1 on AML leukemic stem cells (LSCs). In certain embodiments, the LSCs further express cell membrane markers selected from CD34, CD38, CD123, TIM3, CD25, CD32, and CD96.

In some embodiments, the AML is minimal residual disease (MRD). In certain embodiments, the MRD comprises FLT3-ITD ((Fms-like tyrosine kinase 3)-intragenic tandem duplication (ITD)), NPM1 (nucleophosmin 1), DNMT3A (DNA methyltransferase gene DNMT3A), and IDH ( Characterized by the presence or absence of mutations selected from isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2).

In certain embodiments, MDS is MDS with multilineage dysplasia (MDS-MLD), MDS with single lineage dysplasia (MDS-SLD), MDS with ringed sideroblasts (MDS-RS ), MDS with blast proliferation (MDS-EB), MDS with isolated del (5q), and MDS, unclassified MDS (MDS-U). In certain embodiments, the MDS is a primary MDS or a secondary MDS.

In some embodiments, ALL is selected from B-cell acute lymphoblastic leukemia (B-ALL) and T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the MPN is selected from polycythemia vera, essential thrombocythemia (ET), and myelofibrosis. In some embodiments, the non-Hodgkin's lymphoma is selected from B-cell lymphoma and T-cell lymphoma. In some embodiments, the lymphoma is chronic lymphocytic leukemia (CLL), lymphoblastic lymphoma (LPL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), mediastinal primary Cellular B-cell lymphoma (PMBL), follicular lymphoma, mantle cell lymphoma, pilocytic leukemia, plasma cell myeloma (PCM) or multiple myeloma (MM), mature T/NK cell tumors, and histiocytic tumors is selected from In certain embodiments, the cancer expresses CLEC12A.

1 is a representation of a heterodimeric, multispecific antibody, eg, a trispecific binding protein (TriNKET). Each arm may represent either an NKG2D binding domain or a binding domain corresponding to a tumor-associated antigen. In some embodiments, the NKG2D binding domain and tumor-associated antigen binding domain may share a common light chain.

Five exemplary formats of multispecific binding proteins, eg, trispecific binding proteins (TriNKET) are illustrated. As shown in A, either the NKG2D binding domain or the tumor-associated antigen binding domain may be in scFv format (left arm). An antibody comprising a scFv targeting NKG2D, a Fab fragment targeting a tumor-associated antigen, and a heterodimerized antibody constant region is referred to herein as F3-TriNKET. Antibodies comprising scFvs targeting tumor-associated antigens, Fab fragments targeting NKG2D, and heterodimerized antibody constant regions/domains (which bind CD16) are referred to herein as F3'-TriNKET. called (E). As shown in B, both the NKG2D binding domain and the tumor-associated antigen binding domain can be in scFv format. CD are illustrations of antibodies with three antigen binding sites, including two antigen binding sites that bind tumor-associated antigens, and an NKG2D binding site fused to a heterodimerizing antibody constant region. These antibody formats are referred to herein as F4-TriNKET. C illustrates that the two tumor-associated antigen binding sites are in Fab fragment format and the NKG2D binding site is in scFv format. D illustrates that the tumor-associated antigen binding site is in scFv format and the NKG2D binding site is in scFv format. E represents a trispecific antibody (TriNKET) comprising a tumor-targeting scFv, an NKG2D-targeting Fab fragment, and a heterodimerizing antibody constant region/domain (“CD domain”) that binds CD16. . This antibody format is referred to herein as F3'-TriNKET. In certain exemplary multispecific binding proteins, the heterodimerization mutations on the antibody constant regions are K360E and K409W on one constant domain; and Q347R, D399V, and F405T on the opposite constant domain ( shown as a triangular lock-and-key shape in the CD domain). Bold bars between the heavy and light chain variable domains of the Fab fragment represent disulfide bonds. Five exemplary formats of multispecific binding proteins, eg, trispecific binding proteins (TriNKET) are illustrated. As shown in A, either the NKG2D binding domain or the tumor-associated antigen binding domain may be in scFv format (left arm). An antibody comprising a scFv targeting NKG2D, a Fab fragment targeting a tumor-associated antigen, and a heterodimerized antibody constant region is referred to herein as F3-TriNKET. Antibodies comprising scFvs targeting tumor-associated antigens, Fab fragments targeting NKG2D, and heterodimerized antibody constant regions/domains (which bind CD16) are referred to herein as F3'-TriNKET. called (E). As shown in B, both the NKG2D binding domain and the tumor-associated antigen binding domain can be in scFv format. CD are illustrations of antibodies with three antigen binding sites, including two antigen binding sites that bind tumor-associated antigens, and an NKG2D binding site fused to a heterodimerizing antibody constant region. These antibody formats are referred to herein as F4-TriNKET. C illustrates that the two tumor-associated antigen binding sites are in Fab fragment format and the NKG2D binding site is in scFv format. D illustrates that the tumor-associated antigen binding site is in scFv format and the NKG2D binding site is in scFv format. E represents a trispecific antibody (TriNKET) comprising a tumor-targeting scFv, an NKG2D-targeting Fab fragment, and a heterodimerizing antibody constant region/domain (“CD domain”) that binds CD16. . This antibody format is referred to herein as F3'-TriNKET. In certain exemplary multispecific binding proteins, the heterodimerization mutations on the antibody constant regions are K360E and K409W on one constant domain; and Q347R, D399V, and F405T on the opposite constant domain ( shown as a triangular lock-and-key shape in the CD domain). Bold bars between the heavy and light chain variable domains of the Fab fragment represent disulfide bonds. Five exemplary formats of multispecific binding proteins, eg, trispecific binding proteins (TriNKET) are illustrated. As shown in A, either the NKG2D binding domain or the tumor-associated antigen binding domain may be in scFv format (left arm). An antibody comprising a scFv targeting NKG2D, a Fab fragment targeting a tumor-associated antigen, and a heterodimerized antibody constant region is referred to herein as F3-TriNKET. Antibodies comprising scFvs targeting tumor-associated antigens, Fab fragments targeting NKG2D, and heterodimerized antibody constant regions/domains (which bind CD16) are referred to herein as F3'-TriNKET. called (E). As shown in B, both the NKG2D binding domain and the tumor-associated antigen binding domain can be in scFv format. CD are illustrations of antibodies with three antigen binding sites, including two antigen binding sites that bind tumor-associated antigens, and an NKG2D binding site fused to a heterodimerizing antibody constant region. These antibody formats are referred to herein as F4-TriNKET. C illustrates that the two tumor-associated antigen binding sites are in Fab fragment format and the NKG2D binding site is in scFv format. D illustrates that the tumor-associated antigen binding site is in scFv format and the NKG2D binding site is in scFv format. E represents a trispecific antibody (TriNKET) comprising a tumor-targeting scFv, an NKG2D-targeting Fab fragment, and a heterodimerizing antibody constant region/domain (“CD domain”) that binds CD16. . This antibody format is referred to herein as F3'-TriNKET. In certain exemplary multispecific binding proteins, the heterodimerization mutations on the antibody constant regions are K360E and K409W on one constant domain; and Q347R, D399V, and F405T on the opposite constant domain ( shown as a triangular lock-and-key shape in the CD domain). Bold bars between the heavy and light chain variable domains of the Fab fragment represent disulfide bonds. Five exemplary formats of multispecific binding proteins, eg, trispecific binding proteins (TriNKET) are illustrated. As shown in A, either the NKG2D binding domain or the tumor-associated antigen binding domain may be in scFv format (left arm). An antibody comprising a scFv targeting NKG2D, a Fab fragment targeting a tumor-associated antigen, and a heterodimerized antibody constant region is referred to herein as F3-TriNKET. Antibodies comprising scFvs targeting tumor-associated antigens, Fab fragments targeting NKG2D, and heterodimerized antibody constant regions/domains (which bind CD16) are referred to herein as F3'-TriNKET. called (E). As shown in B, both the NKG2D binding domain and the tumor-associated antigen binding domain can be in scFv format. CD are illustrations of antibodies with three antigen binding sites, including two antigen binding sites that bind tumor-associated antigens, and an NKG2D binding site fused to a heterodimerizing antibody constant region. These antibody formats are referred to herein as F4-TriNKET. C illustrates that the two tumor-associated antigen binding sites are in Fab fragment format and the NKG2D binding site is in scFv format. D illustrates that the tumor-associated antigen binding site is in scFv format and the NKG2D binding site is in scFv format. E represents a trispecific antibody (TriNKET) comprising a tumor-targeting scFv, an NKG2D-targeting Fab fragment, and a heterodimerizing antibody constant region/domain (“CD domain”) that binds CD16. . This antibody format is referred to herein as F3'-TriNKET. In certain exemplary multispecific binding proteins, the heterodimerization mutations on the antibody constant regions are K360E and K409W on one constant domain; and Q347R, D399V, and F405T on the opposite constant domain ( shown as a triangular lock-and-key shape in the CD domain). Bold bars between the heavy and light chain variable domains of the Fab fragment represent disulfide bonds. Five exemplary formats of multispecific binding proteins, eg, trispecific binding proteins (TriNKET) are illustrated. As shown in A, either the NKG2D binding domain or the tumor-associated antigen binding domain may be in scFv format (left arm). An antibody comprising a scFv targeting NKG2D, a Fab fragment targeting a tumor-associated antigen, and a heterodimerized antibody constant region is referred to herein as F3-TriNKET. Antibodies comprising scFvs targeting tumor-associated antigens, Fab fragments targeting NKG2D, and heterodimerized antibody constant regions/domains (which bind CD16) are referred to herein as F3'-TriNKET. called (E). As shown in B, both the NKG2D binding domain and the tumor-associated antigen binding domain can be in scFv format. CD are illustrations of antibodies with three antigen binding sites, including two antigen binding sites that bind tumor-associated antigens, and an NKG2D binding site fused to a heterodimerizing antibody constant region. These antibody formats are referred to herein as F4-TriNKET. C illustrates that the two tumor-associated antigen binding sites are in Fab fragment format and the NKG2D binding site is in scFv format. D illustrates that the tumor-associated antigen binding site is in scFv format and the NKG2D binding site is in scFv format. E represents a trispecific antibody (TriNKET) comprising a tumor-targeting scFv, an NKG2D-targeting Fab fragment, and a heterodimerizing antibody constant region/domain (“CD domain”) that binds CD16. . This antibody format is referred to herein as F3'-TriNKET. In certain exemplary multispecific binding proteins, the heterodimerization mutations on the antibody constant regions are K360E and K409W on one constant domain; and Q347R, D399V, and F405T on the opposite constant domain ( shown as a triangular lock-and-key shape in the CD domain). Bold bars between the heavy and light chain variable domains of the Fab fragment represent disulfide bonds.

A representation of the Triomab form of TriNKET, a trifunctional bispecific antibody that maintains an IgG-like shape. This chimera consists of two half-antibodies, each with one light chain and one heavy chain, derived from the two parental antibodies. The triomab form may be a heterodimeric construct comprising 1/2 rat antibody and 1/2 mouse antibody.

A representation of the KiH Common Light Chain form of TriNKET, which includes knob-in-to-hole (KIH) technology. KiH is a heterodimer comprising two Fab fragments that bind targets 1 and 2, and an Fc stabilized by heterodimerization mutations. A KiH format TriNKET can be a heterodimeric construct with two Fab fragments that bind target 1 and target 2, comprising two different heavy chains and a common light chain that pairs with both heavy chains.

A representation of a dual variable domain immunoglobulin (DVD-Ig™) form of TriNKET, which joins the target binding domains of two monoclonal antibodies via a flexible natural linker to produce a tetravalent IgG-like molecule. do. DVD-Ig™ is a homodimeric construct in which the antigen 2 targeting variable domain is fused to the N-terminus of the antigen 1 targeting Fab fragment variable domain. The DVD-Ig™ format contains normal Fc.

A representation of TriNKET in the orthogonal Fab fragment interface (Ortho-Fab) form, which is a heterodimeric construct comprising two Fab fragments that bind target 1 and target 2 fused to Fc. Light chain (LC)-heavy chain (HC) pairing is ensured by orthogonal interfaces. Heterodimerization is ensured by Fc mutations.

Presentation of TriNKET in a two-in-one (2-in-1) Ig format.

A representation of the ES form of TriNKET, which is a heterodimeric construct comprising two different Fab fragments that bind target 1 and target 2 fused to Fc. Heterodimerization is ensured by electrostatic steering mutations of Fc.

Schematic of Fab arm exchange form of TriNKET: exchanging Fab fragment arms by exchanging the heavy and associated light chains (half-molecules) with heavy-light chain pairs of another molecule to achieve bispecificity. Antibodies that bring about Fab arm-swapped forms (cFae) are heterodimers comprising two Fab fragments that bind targets 1 and 2, and an Fc stabilized by heterodimerization mutations.

FIG. 10 is a representation of the SEED Body form of TriNKET, a heterodimer containing two Fab fragments that bind targets 1 and 2, and an Fc stabilized by heterodimerization mutations.

A representation of the LuZ-Y form of TriNKET that uses a leucine zipper to induce heterodimerization of two different HCs. The LuZ-Y form is a heterodimer containing two different scFabs binding targets 1 and 2 fused to Fc. Heterodimerization is ensured by a leucine zipper motif fused to the C-terminus of Fc.

A representation of the Cov-X-Body form of TriNKET.

AB are representations of the κλ-Body form of TriNKET, which is a heterodimeric construct with two different Fab fragments fused to an Fc stabilized by heterodimerization mutations: antigen. One Fab fragment targeting Antigen 1 contains a kappa LC and a second Fab fragment targeting antigen 2 contains a lambda LC. A is an exemplary representation of one form of κλ-Body. B is an exemplary presentation of another κλ-Body.

An Oasc-Fab heterodimer construct comprising a Fab fragment that binds Target 1 and a scFab that binds Target 2, both fused to an Fc domain. Heterodimerization is ensured by Fc domain mutations.

DuetMab, a heterodimeric construct comprising two different Fab fragments that bind antigens 1 and 2, and an Fc stabilized by heterodimerization mutations. Fab fragments 1 and 2 contain differential SS bridges that ensure correct pairing of light and heavy chains.

CrossmAb, which is a heterodimeric construct with two different Fab fragments that bind targets 1 and 2, and an Fc stabilized by heterodimerization mutations. The CL and CH1 domains and the VH and VL domains are switched, eg CH1 is fused in-line with VL and CL is fused in-line with VH.

Fit-Ig, a homodimeric construct in which the Fab fragment that binds antigen 2 is fused to the N-terminus of the HC of the Fab fragment that binds antigen 1; This construct contains a wild-type Fc.

1 is a series of traces showing the Bio-Layer Interferometry (BLI) profile of antibodies collected from mouse hybridoma supernatants that bind hCLEC12A. 1 is a series of traces showing the Bio-Layer Interferometry (BLI) profile of antibodies collected from mouse hybridoma supernatants that bind hCLEC12A.

19A is a series of sensorgrams showing the SPR profiles of antibodies collected from mouse mAb subclones that bind hCLEC12A (FIG. 19A) and cCLEC12A (FIG. 19B).

FIG. 3 is a line graph showing binding of purified CLEC12A subclones to PL21 AML cell line.

Antibody 16B8 that binds to glycosylated, deglycosylated and desialylated hCLEC12A. C8 and 9F11. 2 is a series of sensorgrams showing the SPR profile of B7.

Antibody 16B8. 2 is a series of sensorgrams showing the SPR profile of TriNKETs derived from C8. Antibody 16B8. 2 is a series of sensorgrams showing the SPR profile of TriNKETs derived from C8. Antibody 16B8. 2 is a series of sensorgrams showing the SPR profile of TriNKETs derived from C8. Antibody 16B8. 2 is a series of sensorgrams showing the SPR profile of TriNKETs derived from C8.

FIG. 3 is a line graph showing the binding of hCLEC12A-targeted TriNKETs F3′-1304, F3′-1295 and control CLEC12A-TriNKETs to the hCLEC12A-expressing cell line RMA-hCLEC12A.

FIG. 3 is a line graph showing NK cell-mediated lysis of CLEC12A-expressing cancer cell line HL60 in the presence of F3'-1295 and control CLEC12A-TriNKET.

1 is a line graph showing differential scanning calorimetry (DSC) analysis of TriNKET F3'-1295 and F3'-1602.

2 is a series of sensorgrams showing the SPR profiles of TriNKET F3'-1295 and F3'-1602. 2 is a series of sensorgrams showing the SPR profiles of TriNKET F3'-1295 and F3'-1602.

hCLEC12A-targeting TriNKET F3′-1295, F3′-1602, and control CLEC12A-TriNKET hCLEC12A-expressing cell line Ba/F3 (A), wild-type Ba/F3 (B), cancer line HL60 (C), and a line graph showing binding to cancer line PL21(D).

28A is a line graph showing NK cell-mediated lysis of CLEC12A-expressing cancer cell lines PL21 (FIG. 28A) and HL60 (FIG. 28B) in the presence of F3'-1295, F3'-1602, and control CLEC12A-TriNKET.

Figure 29B is a line graph showing the isoelectric point (pI) determined by capillary isoelectric focusing (cIEF) of F3'-1295 (Figure 29A) and F3'-1602 (Figure 29B).

Flow cytometry-based polyspecific reagent (PSR) analysis of F3'-1602 TriNKET and F3'-1295 TriNKET.

FIG. 3 is a series of sensorgrams showing the SPR profiles of hF3'-1602 TriNKET and F3-TriNKET AB0010.

FIG. 4 is a line graph showing binding of hF3′-1602 TriNKET and F3-TriNKET AB0010 to hCLEC12A-expressing Ba/F3 (A), cancer line HL60 (B), and wild-type Ba/F3 (C).

Line graph showing NK cell-mediated lysis of CLEC12A-expressing cancer cell line PL21 using KHYG-CD16V cells (A) and primary NK cells (B) in the presence of hF3′-1602 TriNKET and F3-TriNKET AB0010. be.

9F11. into Ba/F3 (A) expressing hCLEC12A and cancer line HL60 (B). FIG. 3 is a line graph showing binding of B7 and hF3′-1602 TriNKETs and TriNKETs derived from F3-TriNKET AB0010.

9F11. FIG. 4 is a line graph showing NK cell-mediated lysis of CLEC12A-expressing cancer cell line HL60 in the presence of B7-derived TriNKETs.

Figure 10 is a line graph showing NK cell-mediated lysis of CLEC12A-expressing cancer cell line HL60 using KHYG-CD16V cells (A) and primary NK cells (B) in the presence of hF3'-1602 TriNKET and AB0192.

Line graph showing NK cell-mediated lysis of CLEC12A-expressing cancer cell lines PL21 (A) and THP-1 (B) in the presence of hF3'-1602 TriNKET and parental mAbs.

FIG. 3 is a line graph showing binding of hF3′-1602 TriNKET to cancer cell lines HL60 (A) and PL21 (B).

Line graph showing binding of TriNKET targeting CLEC12A, parental mAb, and AB0237 NKG2D dead arm variant TriNKET to Ba/F3 (A) expressing hCLEC12A and cancer cell lines HL60 (B) and PL21 (C). is. Line graph showing binding of TriNKET targeting CLEC12A, parental mAb, and AB0237 NKG2D dead arm variant TriNKET to Ba/F3 (A) expressing hCLEC12A and cancer cell lines HL60 (B) and PL21 (C). is. Line graph showing binding of TriNKET targeting CLEC12A, parental mAb, and AB0237 NKG2D dead arm variant TriNKET to Ba/F3 (A) expressing hCLEC12A and cancer cell lines HL60 (B) and PL21 (C). is.

Bar graphs showing surface retention of CLEC12A-targeted TriNKET and parental mAbs on cell lines PL21 (Fig. 40A) and HL60 (Fig. 40B).

Figure 41B is a line graph showing NK cell-mediated lysis of CLEC12A-expressing cancer cell lines PL21 (Fig. 41A) and HL60 (Fig. 41B) in the presence of hF3'-1602 TriNKET, AB0192 and parental mAb.

FIG. 42B is a line graph showing NK cell-mediated lysis of CLEC12A-expressing cancer cell lines PL21 (FIG. 42A) and HL60 (FIG. 42B) in the presence of hF3'-1602 TriNKET.

FIG. 4 is a line graph showing CD8-T cell-mediated lysis of CLEC12A-expressing cancer cell line PL21 in the presence of hF3'-1602 TriNKET, parental mAb, or its NKG2D dead variant.

A series of histograms showing binding of hF3'-1602 TriNKET (grey), parental mAb (white), and control IgG (black) to blood cells.

10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to recombinant human CD64. 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to recombinant human CD64.

10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to CD32aH131. 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to CD32aH131.

FIG. 3 is a series of sensorgrams showing the SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to the human CD32A R131 allele (FCγRIIa R131). FIG. 3 is a series of sensorgrams showing the SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to the human CD32A R131 allele (FCγRIIa R131).

10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD16a high affinity allele V158 (FCγRIIIa V158).

10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD16a low affinity allele F158 (FCγRIIIa F158). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD16a low affinity allele F158 (FCγRIIIa F158).

10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD32b (FCγRIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD32b (FCγRIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD32b (FCγRIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD32b (FCγRIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD32b (FCγRIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD32b (FCγRIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD32b (FCγRIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD32b (FCγRIIb).

10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD16b (FCγRIIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD16b (FCγRIIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD16b (FCγRIIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD16b (FCγRIIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD16b (FCγRIIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD16b (FCγRIIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD16b (FCγRIIIb). 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET and trastuzumab control binding to human CD16b (FCγRIIIb).

10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET binding to cynomolgus monkey (cyno) CD16 and trastuzumab control. 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET binding to cynomolgus monkey (cyno) CD16 and trastuzumab control. 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET binding to cynomolgus monkey (cyno) CD16 and trastuzumab control. 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET binding to cynomolgus monkey (cyno) CD16 and trastuzumab control.

10 is a series of sensorgrams showing the SPR profiles of hF3'-1602 TriNKET binding to human FcRn and trastuzumab control measured at pH 6.0. 10 is a series of sensorgrams showing the SPR profiles of hF3'-1602 TriNKET binding to human FcRn and trastuzumab control measured at pH 6.0. 10 is a series of sensorgrams showing the SPR profiles of hF3'-1602 TriNKET binding to human FcRn and trastuzumab control measured at pH 6.0. 10 is a series of sensorgrams showing the SPR profiles of hF3'-1602 TriNKET binding to human FcRn and trastuzumab control measured at pH 6.0.

10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET binding to cynomolgus monkey (cyno) FcRn and trastuzumab control measured at pH 6.0. 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET binding to cynomolgus monkey (cyno) FcRn and trastuzumab control measured at pH 6.0. 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET binding to cynomolgus monkey (cyno) FcRn and trastuzumab control measured at pH 6.0. 10 is a series of sensorgrams showing SPR profiles of hF3'-1602 TriNKET binding to cynomolgus monkey (cyno) FcRn and trastuzumab control measured at pH 6.0.

FIG. 4 is a series of sensorgrams showing the SPR profile of hF3'-1602 TriNKET binding to human CLEC12A. FIG. 4 is a series of sensorgrams showing the SPR profile of hF3'-1602 TriNKET binding to human CLEC12A.

FIG. 3 is a series of sensorgrams showing the SPR profile of hF3'-1602 TriNKET binding to human NKG2D. FIG. 3 is a series of sensorgrams showing the SPR profile of hF3'-1602 TriNKET binding to human NKG2D.

FIG. 4 is a series of sensorgrams showing the SPR profile of hF3′-1602 TriNKET binding to recombinant cynomolgus monkey (cyno) NKG2D (cNKG2D) extracellular domain (ECD). FIG. 4 is a series of sensorgrams showing the SPR profile of hF3′-1602 TriNKET binding to recombinant cynomolgus monkey (cyno) NKG2D (cNKG2D) extracellular domain (ECD).

FIG. 10 is a line graph showing target binding as determined by SPR for occupancy of one TriNKET arm in binding of the other TriNKET arm.

FIG. 10 is a line graph showing target binding as determined by SPR for the synergistic effect of the NKG2D TriNKET arm and CD16a. FIG. 10 is a line graph showing target binding as determined by SPR for the synergistic effect of the NKG2D TriNKET arm and CD16a.

hCLEC12A-TriNKET F3′-1295, F3 against hCLEC12A-expressing cell line Ba/F3 (FIG. 60A), wild-type Ba/F3 (FIG. 60B), cancer line HL60 (FIG. 60C), and cancer line PL21 (FIG. 60D) FIG. 10 is a line graph showing the binding of '-1602, and control CLEC12A-TriNKET. hCLEC12A-TriNKET F3′-1295, F3 against hCLEC12A-expressing cell line Ba/F3 (FIG. 60A), wild-type Ba/F3 (FIG. 60B), cancer line HL60 (FIG. 60C), and cancer line PL21 (FIG. 60D) FIG. 10 is a line graph showing the binding of '-1602, and control CLEC12A-TriNKET. hCLEC12A-TriNKET F3′-1295, F3 against hCLEC12A-expressing cell line Ba/F3 (FIG. 60A), wild-type Ba/F3 (FIG. 60B), cancer line HL60 (FIG. 60C), and cancer line PL21 (FIG. 60D) FIG. 10 is a line graph showing the binding of '-1602, and control CLEC12A-TriNKET.

Flow cytometry-based polyspecific reagent (PSR) analysis of F3'-1602 TriNKET. Flow cytometry-based polyspecific reagent (PSR) analysis of F3'-1602 TriNKET.

Bar graph showing the relative binding (Z-score) of F3'-1602 TriNKET at 1 to human CLEC12A compared to the entire human proteome microarray.

A model of the hydrophobic patch of the hF3'-1602 TriNKET CLEC12A binding arm is shown.

Bar graphs based on models of CDR length, surface hydrophobicity, and surface charge of the hF3'-1602 TriNKET CLEC12A binding arm. Bar graphs based on models of CDR length, surface hydrophobicity, and surface charge of the hF3'-1602 TriNKET CLEC12A binding arm. Bar graphs based on models of CDR length, surface hydrophobicity, and surface charge of the hF3'-1602 TriNKET CLEC12A binding arm.

A model of the hydrophobic patch of the hF3'-1602 TriNKET NKG2D binding arm is shown.

Bar graphs based on models of CDR length, surface hydrophobicity, and surface charge of the hF3'-1602 TriNKET NKG2D binding arm. Bar graphs based on models of CDR length, surface hydrophobicity, and surface charge of the hF3'-1602 TriNKET NKG2D binding arm. Bar graphs based on models of CDR length, surface hydrophobicity, and surface charge of the hF3'-1602 TriNKET NKG2D binding arm.

FIG. 2 is a line graph showing hydrophobic interaction chromatography (HIC) analysis of hF3'-1602 TriNKET.

Figure 10 is a line graph showing pI determined by cIEF for hF3'-1602 TriNKET.

FIG. 4 is a series of flow cytometry plots showing sequence liability-correcting variants of hF3'-1602 binding to hCLEC12A. FIG. 4 is a series of flow cytometry plots showing sequence liability-correcting variants of hF3'-1602 binding to hCLEC12A. FIG. 4 is a series of flow cytometry plots showing sequence liability-correcting variants of hF3'-1602 binding to hCLEC12A. FIG. 4 is a series of flow cytometry plots showing sequence liability-correcting variants of hF3'-1602 binding to hCLEC12A. FIG. 4 is a series of flow cytometry plots showing sequence liability-correcting variants of hF3'-1602 binding to hCLEC12A.

FIG. 3 is a line graph showing NK cell-mediated lysis of CLEC12A-expressing cancer cell line PL21 in the presence of hF3′-1602 TriNKET and liability-correcting variants.

A series of colored sensorgrams showing binding of hCLEC12A at 3-fold serial dilutions (300 nM to 0.41 nM) to F3'-1602 in HST formulations. Black overlays indicate a 1:1 kinetic fit of the raw data.

FIG. 4 is a series of graphs showing binding of F3′-1602 to hNKG2D in HST formulations. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values. FIG. 4 is a series of graphs showing binding of F3′-1602 to hNKG2D in HST formulations. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values.

FIG. 4 is a series of colored sensorgrams showing binding of F3'-1602 to hCD16aV158 in HST formulations.

FIG. 10 is a graph showing the percentage lysis of PL21 cells by NKG2D and CD16a-expressing KHYG-1-CD16a cells in the presence of stressed and control samples of F3'-1602 in HST formulations.

2 is a series of extracted ion chromatograms of CDRH3 peptides in control and stressed F3'-1602.

FIG. 4 is a series of colored sensorgrams showing binding of hCLEC12A to F3'-1602 in phosphate/citrate/NaCl formulations. Black overlays indicate a 1:1 kinetic fit of the raw data.

FIG. 4 is a series of graphs showing binding of F3′-1602 in phosphate/citrate/NaCl formulations to hNKG2D. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values. FIG. 4 is a series of graphs showing binding of F3′-1602 in phosphate/citrate/NaCl formulations to hNKG2D. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values.

FIG. 4 is a series of colored sensorgrams showing F3'-1602 binding to hCD16a V158 in phosphate/citrate/NaCl formulations.

FIG. 10 is a graph showing the percentage lysis of PL21 cells by NKG2D and CD16a-expressing KHYG-1-CD16a cells in the presence of stressed and control samples of F3′-1602 in phosphate/citrate/NaCl formulations.

4 is a series of colored sensorgrams showing the binding of 3-fold serial dilutions (300 nM to 0.41 nM) of hCLEC12A to F3'-1602 after 4 weeks of incubation with CST formulations at 40°C. Black overlays indicate a 1:1 kinetic fit of the raw data.

FIG. 4 is a series of colored sensorgrams showing that 3-fold serial dilutions (300 nM to 0.41 nM) of hCLEC12A bind to F3′-1602 after 4 weeks of incubation with CST formulations at 2-8° C. and 25° C. FIG. Black overlays indicate a 1:1 kinetic fit of the raw data. FIG. 4 is a series of colored sensorgrams showing that 3-fold serial dilutions (300 nM to 0.41 nM) of hCLEC12A bind to F3′-1602 after 4 weeks of incubation with CST formulations at 2-8° C. and 25° C. FIG. Black overlays indicate a 1:1 kinetic fit of the raw data.

FIG. 4 is a series of graphs showing binding of F3′-1602 to hNKG2D in CST formulations. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values. FIG. 4 is a series of graphs showing binding of F3′-1602 to hNKG2D in CST formulations. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values.

FIG. 4 is a series of colored sensorgrams showing binding of F3'-1602 to hCD16aV158 in CST formulations.

FIG. 10 is a graph showing the percentage lysis of PL21 cells by NKG2D- and CD16a-expressing KHYG-1-CD16a cells in the presence of stressed and control samples of F3'-1602 in CST formulations.

cIEF profile of F3'-1602 after incubation under pH 5.0 stress.

cIEF profile of F3'-1602 after incubation under pH 8.0 stress.

A series of colored sensorgrams showing binding of hCLEC12A at 3-fold serial dilutions (300 nM to 0.41 nM) to F3'-1602 after pH 5 stress. Black overlays indicate a 1:1 kinetic fit of the raw data.

A series of colored sensorgrams showing binding of hCLEC12A at 3-fold serial dilutions (300 nM to 0.41 nM) to F3'-1602 after pH 8 stress. Black overlays indicate a 1:1 kinetic fit of the raw data.

FIG. 4 is a series of graphs showing f3′-1602 binding to hNKG2D after pH 5 stress. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values. FIG. 4 is a series of graphs showing f3′-1602 binding to hNKG2D after pH 5 stress. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values.

FIG. 4 is a series of graphs showing binding of F3′-1602 to hNKG2D after pH 8 stress. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values. FIG. 4 is a series of graphs showing binding of F3′-1602 to hNKG2D after pH 8 stress. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values.

Graph showing the percentage of PL21 cell lysis by NKG2D and CD16a expressing KHYG-1-CD16a cells in the presence of F3'-1602 after pH5 stress.

FIG. 10 is a graph showing the percentage of PL21 cell lysis by NKG2D and CD16a expressing KHYG-1-CD16a cells in the presence of F3′-1602 after pH 8 stress.

FIG. 4 is a series of colored sensorgrams showing binding of hCLEC12A to F3'-1602 after forced oxidative stress. Black overlays indicate a 1:1 kinetic fit of the raw data.

FIG. 4 is a series of graphs showing binding of F3′-1602 to hNKG2D after forced oxidative stress. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values. FIG. 4 is a series of graphs showing binding of F3′-1602 to hNKG2D after forced oxidative stress. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values.

Graph showing the percentage of PL21 cell lysis by NKG2D and CD16a expressing KHYG-1-CD16a cells in the presence of F3'-1602 after forced oxidative stress.

FIG. 4 is a series of colored sensorgrams showing binding of hCLEC12A to F3'-1602 after low pH holding. Black overlays indicate a 1:1 kinetic fit of the raw data.

FIG. 4 is a series of graphs showing binding of F3′-1602 to HNKG2D after low pH holding. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values. FIG. 4 is a series of graphs showing binding of F3′-1602 to HNKG2D after low pH holding. Top panel is a colored sensorgram representing the raw data of F3′-1602 injected into captured mFc-hNKG2D (9.77 nM-5 μM). Black overlays indicate a 1:1 kinetic fit of the raw data. The bottom panel represents the steady-state response measurements plotted against the corresponding analyte concentrations. The blue line represents steady-state _KD values.

A series of colored sensorgrams showing binding of F3'-1602 to hCD16a V158 after low pH hold.

Graph showing the percentage of PL21 cell lysis by NKG2D and CD16a expressing KHYG-1-CD16a cells in the presence of F3'-1602 after low pH hold.

Figure 10 is a graph showing the solubility of F3'-1602 in 10 mM sodium acetate buffer pH 5.0 in PEG-6000.

FIG. 3 is a graph showing the percentage of F3'-1602 monomer content as a function of concentration.

Percent lysis of PL-21 cells by NKG2D and CD16a expressing KHYG-1-CD16a cells in the presence of TriNKETF3′-1602 in F3′ format (FIG. 102A) and TriNKET AB0010 in F3 format (FIG. 102B) after heat stress conditions. is a graph showing

1 is a schematic of yeast display affinity maturation library construction. A: Replacement of pET1596 scFvCDRH3 with BamHI restriction enzyme site. B: Insertion of CDRH3 mutation library oligos at the previously introduced BamHI restriction site. C: Electroporation of BamHI linearized plasmids and library oligos into yeast followed by homologous recombination in yeast. 1 is a schematic of yeast display affinity maturation library construction. A: Replacement of pET1596 scFvCDRH3 with BamHI restriction enzyme site. B: Insertion of CDRH3 mutation library oligos at the previously introduced BamHI restriction site. C: Electroporation of BamHI linearized plasmids and library oligos into yeast followed by homologous recombination in yeast. 1 is a schematic of yeast display affinity maturation library construction. A: Replacement of pET1596 scFvCDRH3 with BamHI restriction enzyme site. B: Insertion of CDRH3 mutation library oligos at the previously introduced BamHI restriction site. C: Electroporation of BamHI linearized plasmids and library oligos into yeast followed by homologous recombination in yeast.

Shows the discovery process from mouse immunization to AB0089. The top panel presents the results of CLEC12A binding by surface plasmon resonance (SPR). Bottom panel shows binding to CLEC12A+ PL-21 AML cancer cells. Binding affinities and EC50s for each variant are indicated. BM, back mutation; Shows the discovery process from mouse immunization to AB0089. The top panel presents the results of CLEC12A binding by surface plasmon resonance (SPR). Bottom panel shows binding to CLEC12A+ PL-21 AML cancer cells. Binding affinities and EC50s for each variant are indicated. BM, back mutation; Shows the discovery process from mouse immunization to AB0089. The top panel presents the results of CLEC12A binding by surface plasmon resonance (SPR). Bottom panel shows binding to CLEC12A+ PL-21 AML cancer cells. Binding affinities and EC50s for each variant are indicated. BM, back mutation; Shows the discovery process from mouse immunization to AB0089. The top panel presents the results of CLEC12A binding by surface plasmon resonance (SPR). Bottom panel shows binding to CLEC12A+ PL-21 AML cancer cells. Binding affinities and EC50s for each variant are indicated. BM, back mutation;

FACS analysis of CDRH3 yeast library 17 is shown. First round of selection of affinity matured yeast display library sorted with 1 nM CLEC12A-His (A). Second round of sorted selection with 1 nM CLEC12A-His (B). Third round of sorted selection with 0.1 nM CLEC12A-His (C). Binding of library 17-R3 displayed in yeast cells to 1 nM CLEC12A (D). Binding of the original clone pET1596 displayed in yeast cells with 10 nM CLEC12A (E). FACS analysis of CDRH3 yeast library 17 is shown. First round of selection of affinity matured yeast display library sorted with 1 nM CLEC12A-His (A). Second round of sorted selection with 1 nM CLEC12A-His (B). Third round of sorted selection with 0.1 nM CLEC12A-His (C). Binding of library 17-R3 displayed in yeast cells to 1 nM CLEC12A (D). Binding of the original clone pET1596 displayed in yeast cells with 10 nM CLEC12A (E). FACS analysis of CDRH3 yeast library 17 is shown. First round of selection of affinity matured yeast display library sorted with 1 nM CLEC12A-His (A). Second round of sorted selection with 1 nM CLEC12A-His (B). Third round of sorted selection with 0.1 nM CLEC12A-His (C). Binding of library 17-R3 displayed in yeast cells to 1 nM CLEC12A (D). Binding of the original clone pET1596 displayed in yeast cells with 10 nM CLEC12A (E).

FACS analysis of CDRH2 yeast library 30 is shown. Starting yeast display library (GDYGDSLDY (SEQ ID NO: 322) CDRH3) generated by randomizing CDRH2 constructed on the background of the best clones identified from library 17 (A). Sorting was performed with 1 nM CLEC12A-His in both rounds. Flow cytometry analysis after round 1 (B) and round 2 (C) selection with CLEC12A-His, respectively. Binding of pET1596 presented to yeast cells with 10 nM CLEC12A-His (D). FACS analysis of CDRH2 yeast library 30 is shown. Starting yeast display library (GDYGDSLDY (SEQ ID NO: 322) CDRH3) generated by randomizing CDRH2 constructed on the background of the best clones identified from library 17 (A). Sorting was performed with 1 nM CLEC12A-His in both rounds. Flow cytometry analysis after round 1 (B) and round 2 (C) selection with CLEC12A-His, respectively. Binding of pET1596 presented to yeast cells with 10 nM CLEC12A-His (D).

Comparison of improved affinity clones identified from Library 30 (A) with pET1596 (B) and AB0237 (C). Comparison of improved affinity clones identified from Library 30 (A) with pET1596 (B) and AB0237 (C). Comparison of improved affinity clones identified from Library 30 (A) with pET1596 (B) and AB0237 (C).

Figure 2 is a schematic of AB0089; Heterodimerization mutations and engineered interchain disulfide bridges ensure efficient chain pairing of IgG1Fc. The scFv was humanized and sequence liability corrected CLEC12A mAb16B8. Derived from C8. scFv are stabilized by an engineered disulfide bridge between VH and VL.

X-ray crystal structure of the EW-RVTFc heterodimer. Superposition of the overall structure of the EW-RVT Fc heterodimer with the human wild-type (WT) IgG1 Fc homodimer (PDB ID: 3AVE) (Figure 109A). Magnification of the paired region of W-VT interaction (FIG. 109B). Magnification of the paired region of the ER interaction (Fig. 109C).

The crystal structure of the EW-RVTS-S heterodimeric Fc is shown with an enlarged view of the asymmetric disulfide bond formed between residues 349C (CH3A) and 354C (CH3B).

Ribbon diagram models of CLEC12A-binding scFv in three different orientations (upper panel) and corresponding surface charge distributions in the same orientation (lower panel) are shown. Three orientations are shown: both frontal (left panel: front view; middle panel: back view) and antigen-engaging surface (right panel: top view).

Figure 3 shows analysis of CDR lengths and surface hydrophobic patches of the CLEC12A targeting arm of AB0089. The arrow points to the line where the CLEC12A-targeting scFv of AB0089 lies relative to the advanced clinical stage mAb. The inner two dashed lines indicate the 2SD (>95% of reference molecules within this region). The outermost two dashed lines indicate the 3SD (>99.7% of reference molecules within this region).

Analysis of patches containing positive (left) and negative charges (middle) and the symmetry of these charges around the CDR regions (right) is shown. Surface charge and charge symmetry analysis of the CLEC12A targeting arm of AB0089. The arrow points to the line where the CLEC12A-targeting scFv of AB0089 is positioned relative to the advanced clinical stage mAb. Each plot has two dashed lines - one narrower and the other closer to a solid line. A dashed line closer to the solid line indicates 2SD (>95% of reference molecules in this region), while a dashed line closer to the solid line indicates 3SD (>99.7% of reference molecules in this region). Analysis of patches containing positive (left) and negative charges (middle) and the symmetry of these charges around the CDR regions (right) is shown. Surface charge and charge symmetry analysis of the CLEC12A targeting arm of AB0089. The arrow points to the line where the CLEC12A-targeting scFv of AB0089 is positioned relative to the advanced clinical stage mAb. Each plot has two dashed lines - one narrower and the other closer to a solid line. A dashed line closer to the solid line indicates 2SD (>95% of reference molecules in this region), while a dashed line closer to the solid line indicates 3SD (>99.7% of reference molecules in this region).

NKG2D binding Fabs with three different orientation ribbon views are shown. Both front (left panel: front view, middle panel: back view) and antigen-binding surface (right panel: top view).

Figure 3 shows analysis of CDR lengths and surface hydrophobic patches of the NKG2D targeting arm of AB0089. The arrow points to the line where the NKG2D targeting arm of AB0089 lies relative to the advanced clinical stage mAb. The inner two dashed lines indicate the 2SD (>95% of reference molecules within this region). The outermost two dashed lines indicate the 3SD (>99.7% of reference molecules within this region).

Figure 3 shows surface charge and hydrophobicity analyzes of the NKG2D targeting arm of AB0089. Arrows indicate the location of the NKG2D targeting arm of AB0089 with reference to a database of 377 late-stage therapeutic antibodies. Each plot has two dashed lines - one narrower and the other closer to a solid line. A dashed line close to the solid line indicates 2SD (>95% of reference molecules in this region), while a dashed line even closer to the solid line indicates 3SD (>99.7% of reference molecules in this region). Figure 3 shows surface charge and hydrophobicity analyzes of the NKG2D targeting arm of AB0089. Arrows indicate the location of the NKG2D targeting arm of AB0089 with reference to a database of 377 late-stage therapeutic antibodies. Each plot has two dashed lines - one narrower and the other closer to a solid line. A dashed line close to the solid line indicates 2SD (>95% of reference molecules in this region), while a dashed line even closer to the solid line indicates 3SD (>99.7% of reference molecules in this region).

Figure 3 shows the human acceptor framework used in AB0089 compared to other clinical stage therapeutic mAbs. Benchmarking the framework used in AB0089 against human germline frequently used in clinical-stage antibody databases containing 400+ mAbs from Phase I+. Figure 3 shows the human acceptor framework used in AB0089 compared to other clinical stage therapeutic mAbs. Benchmarking the framework used in AB0089 against human germline frequently used in clinical-stage antibody databases containing 400+ mAbs from Phase I+.

A cartoon representation of AB0089 showing the chain composition of the molecule (A) and the position of chains H, S, and L of AB0089 on the Treg-adjusted EpiMatrix protein immunogenicity scale (B). A cartoon representation of AB0089 showing the chain composition of the molecule (A) and the position of chains H, S, and L of AB0089 on the Treg-adjusted EpiMatrix protein immunogenicity scale (B).

FIG. 13 shows a flow diagram of the TriNKET purification process.

Shown is a MabSelect protein A capture elution chromatogram.

SEC analysis of pH adjusted and filtered protein A eluate for IEX loading.

SDS-PAGE analysis of AB0089 Protein A load, flow-through, and eluate. Based on absorbance readings, 15 μl was loaded in the “Protein A Load” and “Protein A FT” lanes and 2 μg was loaded in the “Protein A Elution Lane”. A major band approximately 120 kDa corresponds to AB0089.

Poros XS cation exchange chromatogram is shown.

SDS-PAGE analysis of load and elution fractions of AB0089 CIEX is shown. Based on absorbance readings, lanes contain approximately 2.0 μg of protein. The major band approximately 120 kDa is AB0089.

Reducing (“R”) and non-reducing (“NR”) SDS-PAGE of AB0089 lot AB0089-002 are shown. The three strands of the molecule (S, H, and L) are labeled in the R lane. NR lane F3' refers to AB0089.

SEC analysis of purified AB0089 lot AB0089-002 is shown, showing 100% monomer as determined by integrated peak area.

Shown is intact mass analysis of AB0089 lot AB0089-002. LC-MS analysis of intact AB0089 showed an observed mass (126,130.0 Da) in good agreement with a theoretical mass of 126,129.1 Da.

CE-SDS analysis of AB0089, R (left panel) and CE-SDS analysis of AB0089, NR (right panel) are shown. CE-SDS analysis of AB0089, R (left panel) and CE-SDS analysis of AB0089, NR (right panel) are shown.

Figure 2 shows the charge profile of AB0089 by cIEF, which shows that AB0089 exhibited a major peak at pI of 8.52±0.0.

Figure 2 shows the chromatogram of hydrophobicity analysis of AB0089 by HIC, which shows that AB0089 is very pure and homogeneous.

Figure 3 shows thermal stability of AB0089 assessed by DSC analysis in three different formulations: PBS pH 7.4 (A), HST pH 6.0 (A), and CST pH 7.0 (A).

Figure 3 shows the predicted disulfide map of AB0089.

Extracted ion chromatograms (XIC) of engineered disulfide pairs of scFv (non-reduced and reduced) and the strongest charge states of the peptide pairs are shown.

Identification of CH3-CH3 intermolecular Fc engineered disulfides.

Figure 3 shows binding of human CLEC12A to AB0089 tested by SPR. Figure 3 shows binding of human CLEC12A to AB0089 tested by SPR.

Figure 2 shows that CLEC12A AB0089 and Tepositamab-based intermolecular binding is consistent with a two-state model.

FACS evaluation of AB0089 binding to isogenic cell lines expressing CLEC12A and to AML cancer cell lines (PL-21 and HL-60).

AB0089 binding to genetic variant CLEC12A (Q244) (middle panel) compared to CLEC12A (K244) WT (left panel) and cyno CLEC12A (cCLEC12A) (right panel). AB0089 binding to genetic variant CLEC12A (Q244) (middle panel) compared to CLEC12A (K244) WT (left panel) and cyno CLEC12A (cCLEC12A) (right panel).

Differentially glycosylated CLEC12A: AB0089 binding to hCLEC12A untreated (left panel), hCLEC12A desialylated (middle panel), and hCLEC12A fully deglycosylated (right panel). Differentially glycosylated CLEC12A: AB0089 binding to hCLEC12A untreated (left panel), hCLEC12A desialylated (middle panel), and hCLEC12A fully deglycosylated (right panel).

Figure 3 shows binding of AB0089 to human NKG2D tested by SPR. Upper panel: colored sensorgrams represent raw data. Black overlay indicates a 1:1 kinetic fit. Bottom panel: Steady-state affinity fit. Data from four independent Biacore channels are shown. Lines represent steady-state KD values. Figure 3 shows binding of AB0089 to human NKG2D tested by SPR. Upper panel: colored sensorgrams represent raw data. Black overlay indicates a 1:1 kinetic fit. Bottom panel: Steady-state affinity fit. Data from four independent Biacore channels are shown. Lines represent steady-state KD values. Figure 3 shows binding of AB0089 to human NKG2D tested by SPR. Upper panel: colored sensorgrams represent raw data. Black overlay indicates a 1:1 kinetic fit. Bottom panel: Steady-state affinity fit. Data from four independent Biacore channels are shown. Lines represent steady-state KD values.

Binding of AB0089 and trastuzumab to recombinant human CD16a (V158). Binding of AB0089 and trastuzumab to recombinant human CD16a (V158). Binding of AB0089 and trastuzumab to recombinant human CD16a (V158).

Binding of AB0089 and trastuzumab to recombinant human CD64 (FcγRI). Binding of AB0089 and trastuzumab to recombinant human CD64 (FcγRI). Binding of AB0089 and trastuzumab to recombinant human CD64 (FcγRI). Binding of AB0089 and trastuzumab to recombinant human CD64 (FcγRI).

Binding of AB0089 and trastuzumab to cynomolgus monkey CD64 (FcγRI). Binding of AB0089 and trastuzumab to cynomolgus monkey CD64 (FcγRI). Binding of AB0089 and trastuzumab to cynomolgus monkey CD64 (FcγRI). Binding of AB0089 and trastuzumab to cynomolgus monkey CD64 (FcγRI).

Binding of AB0089 and trastuzumab to human CD32a H131 (FcγRIIa H131). Binding of AB0089 and trastuzumab to human CD32a H131 (FcγRIIa H131).

Binding of AB0089 and trastuzumab to human CD32a R131 (FcγRIIa R131). Binding of AB0089 and trastuzumab to human CD32a R131 (FcγRIIa R131). Binding of AB0089 and trastuzumab to human CD32a R131 (FcγRIIa R131). Binding of AB0089 and trastuzumab to human CD32a R131 (FcγRIIa R131).

Binding of AB0089 and trastuzumab to recombinant human CD16a F158 (FcγRIIIa F158). Binding of AB0089 and trastuzumab to recombinant human CD16a F158 (FcγRIIIa F158). Binding of AB0089 and trastuzumab to recombinant human CD16a F158 (FcγRIIIa F158). Binding of AB0089 and trastuzumab to recombinant human CD16a F158 (FcγRIIIa F158).

Figure 3 shows binding of AB0089 and trastuzumab to recombinant cynomolgus monkey CD16 (FcγRIII). Figure 3 shows binding of AB0089 and trastuzumab to recombinant cynomolgus monkey CD16 (FcγRIII). Figure 3 shows binding of AB0089 and trastuzumab to recombinant cynomolgus monkey CD16 (FcγRIII). Figure 3 shows binding of AB0089 and trastuzumab to recombinant cynomolgus monkey CD16 (FcγRIII).

Binding of AB0089 and trastuzumab to recombinant human CD32b (FcγRII). Binding of AB0089 and trastuzumab to recombinant human CD32b (FcγRII). Binding of AB0089 and trastuzumab to recombinant human CD32b (FcγRII). Binding of AB0089 and trastuzumab to recombinant human CD32b (FcγRII).

Binding of AB0089 and trastuzumab to recombinant human CD16b (FcγRIIIb). Binding of AB0089 and trastuzumab to recombinant human CD16b (FcγRIIIb). Binding of AB0089 and trastuzumab to recombinant human CD16b (FcγRIIIb). Binding of AB0089 and trastuzumab to recombinant human CD16b (FcγRIIIb).

Binding of AB0089 and trastuzumab to recombinant human FcRn. Binding of AB0089 and trastuzumab to recombinant human FcRn. Binding of AB0089 and trastuzumab to recombinant human FcRn. Binding of AB0089 and trastuzumab to recombinant human FcRn.

Binding of AB0089 and trastuzumab to recombinant cynomolgus monkey FcRn. Binding of AB0089 and trastuzumab to recombinant cynomolgus monkey FcRn. Binding of AB0089 and trastuzumab to recombinant cynomolgus monkey FcRn. Binding of AB0089 and trastuzumab to recombinant cynomolgus monkey FcRn.

We show that when AB0089 simultaneously engages CD16a and NKG2D, strong binding occurs.

AB0089 simultaneous engagement of the binding arms of CLEC12A and NKG2D. AB0089 simultaneous engagement of the binding arms of CLEC12A and NKG2D.

Summary of comparison of binding epitopes of AB0089 relative to AB0237, AB0192 and three CLEC12A binding reference TriNKETs by SPR. The interaction of AB0089 with hCLEC12A was blocked by AB0237 and two control molecules cFAE-A49 CLL1-Merus and cFAE-A49 Tepoditamab, but not by the Genentech-h6E7-based molecule, which , indicating that the AB0089 binding epitope overlaps with Merus-CLL1 and tepositamab.

Figure 3 shows the potency of AB0089 in KHYG-1-CD16aV-mediated cytotoxicity assays using HL60 and PL21 cells.

We show that AB0089 potently enhances primary NK-mediated cytolysis of cancer cells over the parental mAb.

Shown are PSR assays (Figure 157A) and a schematic representation of PSR binding AB0089 compared to an antibody with known PSR binding (rituximab) and an antibody with no known non-specificity (trastuzumab) (Figure 157B). Shown are PSR assays (Figure 157A) and a schematic representation of PSR binding AB0089 compared to an antibody with known PSR binding (rituximab) and an antibody with no known non-specificity (trastuzumab) (Figure 157B). Shown are PSR assays (Figure 157A) and a schematic representation of PSR binding AB0089 compared to an antibody with known PSR binding (rituximab) and an antibody with no known non-specificity (trastuzumab) (Figure 157B).

Shown is the relative binding (Z-score) of AB0089 at 1 μg/ml to human CLEC12A compared to the entire human proteome microarray. Plot showing relative binding Z-scores (y-axis) and top 24 identified binding partners of AB0089 (x-axis). hCLEC12a is plotted at position 1 with a Z-score of 150.61.

Figure 10 shows SEC analysis of AB0089 after 4 weeks at pH 6.0 at 40°C in HST compared to control sample, showing that AB0089 exhibited high stability.

Shows CE-SDS analysis of AB0089 after 4 weeks at pH 6.0 at 40° C. in HST compared to control. Non-reduced (NR) (left panel) and reduced (R) (right panel). Shows CE-SDS analysis of AB0089 after 4 weeks at pH 6.0 at 40° C. in HST compared to control. Non-reduced (NR) (left panel) and reduced (R) (right panel).

We show that AB0089 is stable in HST at pH 6.0 after 4 weeks at 40° C. and does not affect binding to hCLEC12A (FIG. 161A), hNKG2D (FIG. 161B), or hCD16aV (FIG. 161C). We show that AB0089 is stable in HST at pH 6.0 after 4 weeks at 40° C. and does not affect binding to hCLEC12A (FIG. 161A), hNKG2D (FIG. 161B), or hCD16aV (FIG. 161C). We show that AB0089 is stable in HST at pH 6.0 after 4 weeks at 40° C. and does not affect binding to hCLEC12A (FIG. 161A), hNKG2D (FIG. 161B), or hCD16aV (FIG. 161C).

Shows AB0089 potency after 4 weeks at 40° C. in HST, pH 6.0.

It shows that AB0089 exhibited enhanced stability after 4 weeks of incubation at CST, pH 7.0, 40° C. compared to the control sample as determined by SEC analysis.

FIG. 4 shows that no fragmentation of AB0089 was observed by CE-SDS after 4 weeks at 40° C. in CST, pH 7.0 compared to control samples. NR (left panel) and R (right panel). FIG. 4 shows that no fragmentation of AB0089 was observed by CE-SDS after 4 weeks at 40° C. in CST, pH 7.0 compared to control samples. NR (left panel) and R (right panel).

AB0089 is stable in CST at pH 7.0 after 4 weeks at 40° C. and does not affect binding to hCLEC12A (FIG. 161A), hNKG2D (FIG. 161B), or hCD16aV (FIG. 161C). AB0089 is stable in CST at pH 7.0 after 4 weeks at 40° C. and does not affect binding to hCLEC12A (FIG. 161A), hNKG2D (FIG. 161B), or hCD16aV (FIG. 161C). AB0089 is stable in CST at pH 7.0 after 4 weeks at 40° C. and does not affect binding to hCLEC12A (FIG. 161A), hNKG2D (FIG. 161B), or hCD16aV (FIG. 161C).

We show that long-term heat stress did not affect the potency of AB0089 after 4 weeks at 40° C. at pH 7.0 in CST compared to controls.

Extracted ion chromatogram of a tryptic peptide containing CDRH3 of the CLEC12a targeting arm of AB0089, in which no evidence of aspartate isomerization of the peptide containing CLEC12A-bound CDRH3 was observed based on manual inspection of peak shape. was shown.

SEC analysis, which shows no difference in monomer content between the oxidized AB0089 after forced oxidation and the control sample.

It shows that no fragmentation of AB0089 was observed by CE-SDS after forced oxidation compared to control samples. NR (left panel) and R (right panel). It shows that no fragmentation of AB0089 was observed by CE-SDS after forced oxidation compared to control samples. NR (left panel) and R (right panel).

Oxidative stress did not significantly affect active protein content or affinity for hCLEC12A (Figure 170A, top panel), hNKG2D (Figure 170B, middle panel), and hCD16Av (Figure 170C, bottom panel). . Oxidative stress did not significantly affect active protein content or affinity for hCLEC12A (Figure 170A, top panel), hNKG2D (Figure 170B, middle panel), and hCD16Av (Figure 170C, bottom panel). . Oxidative stress did not significantly affect active protein content or affinity for hCLEC12A (Figure 170A, top panel), hNKG2D (Figure 170B, middle panel), and hCD16Av (Figure 170C, bottom panel). .

It shows that AB0089 is stable under forced oxidation.

SEC analysis shows that AB0089 is stable under high pH stress (pH 8.0, 40° C., 2 weeks).

CE-SDS analysis showing that AB0089 exhibited low levels of degradation after prolonged high pH stress (pH 8, 40° C., 2 weeks). NR (left panel) and R (right panel). CE-SDS analysis showing that AB0089 exhibited low levels of degradation after prolonged high pH stress (pH 8, 40° C., 2 weeks). NR (left panel) and R (right panel).

cIEF, showing that AB0089 exhibited an acidic shift after prolonged high pH exposure (pH 8, 40° C., 2 weeks).

After prolonged high pH 8.0 stress (pH 8, 40° C., 2 weeks), the amount of active protein or hCLEC12A (FIG. 175A, top panel), hNKG2D (FIG. 175B, middle panel) increased between stressed and control samples. panel), or hCD16aV (FIG. 175C, bottom panel). After prolonged high pH 8.0 stress (pH 8, 40° C., 2 weeks), the amount of active protein or hCLEC12A (FIG. 175A, top panel), hNKG2D (FIG. 175B, middle panel) increased between stressed and control samples. panel), or hCD16aV (FIG. 175C, bottom panel). After prolonged high pH 8.0 stress (pH 8, 40° C., 2 weeks), the amount of active protein or hCLEC12A (FIG. 175A, top panel), hNKG2D (FIG. 175B, middle panel) increased between stressed and control samples. panel), or hCD16aV (FIG. 175C, bottom panel).

It shows that the potency of AB0089 decreased about 2-fold after prolonged high pH stress (pH 8, 40° C., 2 weeks).

SEC analysis shows that AB0089 is highly stable under low pH stress (pH 5.0, 40° C., 2 weeks).

CE-SDS analysis of AB0089 after prolonged low pH stress (pH 5.0 40° C., 2 weeks), low levels of AB0089 degradation detected by non-reduced (left panel) and reduced (right panel) CE-SDS. indicates that CE-SDS analysis of AB0089 after prolonged low pH stress (pH 5.0 40° C., 2 weeks), low levels of AB0089 degradation detected by non-reduced (left panel) and reduced (right panel) CE-SDS. indicates that

cIEF analysis showing that AB0089 exposed to low pH stress exhibits a slight acidic shift in charge profile.

After prolonged low pH stress (pH 5.0, 40° C., 2 weeks), active proteins for hCLEC12A (FIG. 180A, upper panel), hNKG2D (FIG. 180B, middle panel), or hCD16aV (FIG. 180C, lower panel) It shows that there was no significant difference in content or AB0089 affinity. After prolonged low pH stress (pH 5.0, 40° C., 2 weeks), active proteins for hCLEC12A (FIG. 180A, upper panel), hNKG2D (FIG. 180B, middle panel), or hCD16aV (FIG. 180C, lower panel) It shows that there was no significant difference in content or AB0089 affinity. After prolonged low pH stress (pH 5.0, 40° C., 2 weeks), active proteins for hCLEC12A (FIG. 180A, upper panel), hNKG2D (FIG. 180B, middle panel), or hCD16aV (FIG. 180C, lower panel) It shows that there was no significant difference in content or AB0089 affinity.

It shows that the potency of AB0089 remains similar to that of control samples after prolonged low pH stress (pH 5.0 40° C., 2 weeks).

Extracted ion chromatogram of a tryptic peptide containing CDRH3 of the CLEC12a targeting arm of AB0089, which contains CLEC12A-bound CDRH3 after prolonged exposure to low pH (pH 5.0), based on manual inspection of peak shape. No evidence of aspartic acid isomerization in the peptide was shown.

SEC analysis showing that AB0089 is stable after 6 freeze/thaw cycles.

CE-SDS analysis of AB0089 after 6 freeze/thaw cycles showing no loss of purity detected by reduced (right panel) or non-reduced CE-SDS (left panel). CE-SDS analysis of AB0089 after 6 freeze/thaw cycles showing no loss of purity detected by reduced (right panel) or non-reduced CE-SDS (left panel).

SEC analysis shows that AB0089 is stable after agitation stress (300 rpm at room temperature, 3 days in deep well plate). SEC analysis shows that AB0089 is stable after agitation stress (300 rpm at room temperature, 3 days in deep well plate).

SEC analysis shows that AB0089 allowed concentration above 175 mg/ml with minimal percent loss of monomer.

Figure 10 is a graph showing that AB0089 can be corrected in concentration (up to 178 mg/ml) without loss of monomer.

SEC analysis of Protein A eluate before and after low pH hold, showing that AB0089 is very stable at the low pH hold/virus inactivation conditions used in the manufacture of biologics.

It shows that the charge profile of AB0089 remains unchanged after low pH holding.

Figure 2 shows that the affinity of AB0089 for hCLEC12a, hNKG2D and CD16aV was not affected by low pH holding. Figure 2 shows that the affinity of AB0089 for hCLEC12a, hNKG2D and CD16aV was not affected by low pH holding. Figure 2 shows that the affinity of AB0089 for hCLEC12a, hNKG2D and CD16aV was not affected by low pH holding.

It shows that the potency of AB0089 did not change after the low pH holding step.

SEC analysis showing that no change in % monomer profile was observed after low pH hold. SEC analysis showing that no change in % monomer profile was observed after low pH hold.

It shows that low pH holding did not significantly affect the charge profile of AB0089.

We show that AB0089 potently enhances human NK cytotoxicity of PL21 AML cells. Purified human NK cells were rested overnight and co-cultured with labeled PL21 target cells for the DELFIA assay the next day. Dose titrations of CLEC12A TriNKET AB0089 or the parental mAb AB0305 were prepared starting at 10 nM and added to co-cultures. The assay was performed using a total of 6 healthy NK cell donors. Shown are representative results. Specific lysis was plotted against test substance concentration and the data were fitted to a four parameter non-linear regression model to generate potency values. Data points represent mean ± standard deviation.

We show that AB0192 potently enhances human NK cytotoxicity of HL60AML cells. Purified human NK cells were rested overnight and co-cultured with labeled HL60 target cells for the DELFIA assay the next day. Dose titrations of CLEC12A TriNKET AB0089 or the parental mAb AB0305 were prepared starting at 10 nM and added to co-cultures. The assay was performed using a total of 4 healthy NK cell donors. Shown are representative results. Specific lysis was plotted against test substance concentration and the data were fitted to a four parameter non-linear regression model to generate potency values. Data points represent mean ± standard deviation.

We show that AB0192 potently enhances human NK cytotoxicity of PL21 AML cells. Purified human NK cells were rested overnight and co-cultured with labeled PL21 target cells for the DELFIA assay the next day. Dose titrations of CLEC12A TriNKET AB0192 or parental mAb AB0306 were prepared starting at 10 nM and added to co-cultures. The assay was performed using a total of 6 healthy NK cell donors. Shown are representative results. Specific lysis was plotted against test substance concentration and the data were fitted to a four parameter non-linear regression model to generate potency values. Data points represent mean ± standard deviation.

We show that AB0192 potently enhances human NK cytotoxicity of HL60 AML cells. Purified human NK cells were rested overnight and co-cultured with labeled HL60 target cells for the DELFIA assay the next day. Dose titrations of CLEC12A TriNKET AB0192 or parental mAb AB0306 were prepared starting at 10 nM and added to co-cultures. The assay was performed using a total of 4 healthy NK cell donors. Shown are representative results. Specific lysis was plotted against test substance concentration and the data were fitted to a four parameter non-linear regression model to generate potency values. Data points represent mean ± standard deviation.

It shows that AB0089 activity is not affected by the presence of soluble MIC-A. Purified human NK cells were rested overnight and prepared the next day in co-culture wells with DELFIA-labeled PL21 target cells. Dose titrations of AB0089 and AB0305 were prepared and soluble MIC-A-Fc was added to each test article titration series at 20 ng/mL. Dose escalation was fit with a four-parameter non-linear regression model. Data points represent mean ± standard deviation.

AB0089 has been shown to induce IFNγ production and degranulation by human NK cells on PL21 AML cells. Frozen PBMCs were thawed and co-cultured with PL21 target cells in the presence of dose titrations of AB0089, AB0305, or F3'-palivizumab. After incubation, cells were prepared for FACS analysis of CD107a degranulation and intracellular accumulation of IFNγ. Figures shown are representative of the three PBMC donors tested. The percentage of IFNγ + CD107 + NK cells induced was plotted against concentration and the data were fit to a four-parameter non-linear regression model to generate potency values. Data points represent mean ± standard deviation.

AB0089 has been shown to induce IFNγ production and degranulation by human NK cells on HL60 AML cells. Frozen PBMCs were thawed and co-cultured with HL60 target cells in the presence of dose titrations of AB0089, AB0305, or F3'-palivizumab. After incubation, cells were prepared for FACS analysis of CD107a degranulation and intracellular accumulation of IFNγ. Figures shown are representative of the three PBMC donors tested. The percentage of IFNγ + CD107 + NK cells induced was plotted against concentration and the data were fit to a four-parameter non-linear regression model to generate potency values. Data points represent mean ± standard deviation.

Figure 2 shows that CLEC12ATriNKET induces M0-macrophage ADCP activity on PL21 AML cells. In vitro generated M0 macrophages were labeled with cell trace violet and co-cultured with cell trace CFSE-labeled PL21 target cells at an E:T ratio of 8:1. After 2 hours, cells were ready for flow cytometry analysis. A double positive cell trace violet/CFSE cells was considered a phagocytic event. The assay was performed using macrophages derived from three independent donors and the figures shown are representative results. The percentage of target cell phagocytosis was plotted against the concentration of test substance and the data were fitted to a four parameter non-linear regression model to generate potency values. Data points represent mean ± standard deviation.

AB0089 does not stimulate CDC on PL21 AML cells. AB0089 was incubated with BATDA-labeled PL21 cells in cell culture medium containing 5% human serum for 60 minutes. A human IgG1 isotype control served as a negative control, while an anti-MHC class I clone W6/32 was used as a positive control. Specific lysis of target cells was plotted against concentration and the data were fit to a four parameter non-linear regression model to generate potency values. Data points represent mean ± standard deviation.

Binding of CLEC12A-TriNKET to cell lines expressing CLEC12A. Dose-response binding of CLEC12A TriNKET and their parental mAbs was analyzed in Ba/F3 cells overexpressing human CLEC12A (A). Human cancer cell lines (B) HL60 and (C) PL21 with endogenous expression of CLEC12A were used to corroborate the results with transfected cell lines. The figure shown represents the results of three independent experiments summarized in Table 201. Binding of CLEC12A-TriNKET to cell lines expressing CLEC12A. Dose-response binding of CLEC12A TriNKET and their parental mAbs was analyzed in Ba/F3 cells overexpressing human CLEC12A (A). Human cancer cell lines (B) HL60 and (C) PL21 with endogenous expression of CLEC12A were used to corroborate the results with transfected cell lines. The figure shown represents the results of three independent experiments summarized in Table 201. Binding of CLEC12A-TriNKET to cell lines expressing CLEC12A. Dose-response binding of CLEC12A TriNKET and their parental mAbs was analyzed in Ba/F3 cells overexpressing human CLEC12A (A). Human cancer cell lines (B) HL60 and (C) PL21 with endogenous expression of CLEC12A were used to corroborate the results with transfected cell lines. The figure shown represents the results of three independent experiments summarized in Table 201.

FIG. 4 shows CLEC12A surface retention in AML cell lines exposed to CLEC12A TriNKETs. CLEC12A surface retention on (A) HL60 and (B) PL21 cells was measured after exposure to test substances at 40 nM for 2 hours at 37°C. Test substances were used at saturating concentrations and detected using a human IgG Fc specific secondary antibody. The staining intensity after 2 hours at 37° C. was normalized to the staining intensity of the same test substance on cells incubated on ice for 2 hours to derive the percentage of CLEC12A retained on the cell surface. Data bars represent the mean of duplicate wells and error bars represent the standard deviation. The figure shown represents the results of three independent experiments summarized in Table 202.

Shows AB0089 binding normalized to AB0237 after 2 hours at 37°C. Binding of AB0089 and AB0237 was compared in HL60 cells after 2 hours incubation on ice or at 37°C. Histograms represent: (first top to bottom) AB0237 2 hours at 37°C, (second top to bottom) AB0237 on ice for 2 hours, (third top to bottom) AB0089 at 37°C. 2 hours, (4th from top to bottom) AB0089 2 hours on ice. Binding of all test substances was detected with an anti-human IgG Fc fluorophore-conjugated antibody.

AB0089 binds to CLEC12A+ blasts across 10 primary AML patient samples. Primary AML bone marrow and PBMC samples were obtained from commercial suppliers. See Table 3. Samples were prepared for flow cytometric analysis according to the antibody panel in Table 1. Histograms represent staining of AML blasts as follows: #4 from top to bottom = hIgG1 isotype control, #3 from top to bottom = AB0305-CF568, #2 from top to bottom = AB0089-CF568, First from top to bottom = anti-CLEC12 50C1-CF568.

AB0089 binds to CLEC12A+CD34+CD38- cells across 10 primary AML patient samples. Primary AML bone marrow and PBMC samples were obtained from commercial suppliers. See Table 200. Samples were prepared for flow cytometric analysis according to the antibody panel in Table 198. Samples were further gated on the CD34+CD38- subpopulation to enrich for leukemic stem cells. Histograms show staining with CD34+CD38− blasts as follows: 4th top to bottom = hIgG1 isotype control, 3rd top to bottom = AB0305-CF568, 2nd top to bottom = AB0089-CF568, top. 1st down from = anti-CLEC12 50C1-CF568.

Pathways from immunization to AB0192 are shown. Top panel shows the results of CLEC12A binding by SPR. Bottom panel shows binding to CLEC12A+ PL-21 AML cancer cells. Pathways from immunization to AB0192 are shown. Top panel shows the results of CLEC12A binding by SPR. Bottom panel shows binding to CLEC12A+ PL-21 AML cancer cells. Pathways from immunization to AB0192 are shown. Top panel shows the results of CLEC12A binding by SPR. Bottom panel shows binding to CLEC12A+ PL-21 AML cancer cells. Pathways from immunization to AB0192 are shown. Top panel shows the results of CLEC12A binding by SPR. Bottom panel shows binding to CLEC12A+ PL-21 AML cancer cells.

Figure 2 shows a schematic diagram of AB0192. Proprietary heterodimerization mutations and engineered interchain disulfide bridges ensure efficient chain pairing of IgG1Fc. scFv is CLEC12A mAb 9F11. Derived from B7. scFv are stabilized by an engineered disulfide bridge between VH and VL.

The amino acid sequences of the three polypeptide chains that make up AB0192 are shown. Chain S: anti-CLEC12 AscFv-CH2-CH3 (humanized sequence, CDRs bold and not underlined, back mutations bold and underlined, engineered scFv disulfides italic and bold, as well as heterodimerizing Fc mutations. and the engineered CH3 disulfides are non-bold and underlined). Chain H: anti-NKGD VH-CH1-CH2-CH3 (fully human, CDRs are bold and not underlined, Fc mutations for heterodimerization, and engineered CH3 disulfides are underlined instead of bold) is subtracted). Chain L: anti-NKGD VL-CL (fully human, CDRs bold and not underlined).

Figure 3 shows the human acceptor framework used in AB0192 compared to other clinical stage therapeutic mAbs. Benchmarking the framework used in AB0192 against human germline frequently used in clinical-stage antibody databases containing 400+ mAbs from Phase I+. Figure 3 shows the human acceptor framework used in AB0192 compared to other clinical stage therapeutic mAbs. Benchmarking the framework used in AB0192 against human germline frequently used in clinical-stage antibody databases containing 400+ mAbs from Phase I+.

Ribbon diagram models of CLEC12A-binding scFv in three different orientations (upper panels) and corresponding surface charge distributions in the same orientation (lower panels) are shown. The charge distribution of the anti-CLEC12A scFv is polarized ('top view', bottom panel), with negatively charged residues predominantly within CDRH3 and CDRL2. Three orientations are shown: both frontal (left panel: front view; middle panel: back view) and antigen-engaging surface (right panel: top view).

Figure 3 shows analysis of CDR lengths and surface hydrophobic patches of the CLEC12A targeting arm of AB0192. Arrows indicate where the CLEC12A-targeting scFv of AB0192 is positioned relative to advanced clinical stage mAbs. The inner dashed line indicates the 2SD (>95% of reference molecules within this region). The outermost dotted line indicates the 3SD (>99.7% of reference molecules within this region).

Analysis of patches containing positive and negative charges and the symmetry of these charges around the CDR regions is shown. Arrows indicate where the CLEC12A-targeting scFv of AB0192 is positioned relative to advanced clinical stage mAbs. Each plot has two dashed lines - one narrower and the other closer to a solid line. A dashed line close to the solid line indicates 2SD (>95% of reference molecules in this region), while a dashed line even closer to the solid line indicates 3SD (>99.7% of reference molecules in this region). Analysis of patches containing positive and negative charges and the symmetry of these charges around the CDR regions is shown. Arrows indicate where the CLEC12A-targeting scFv of AB0192 is positioned relative to advanced clinical stage mAbs. Each plot has two dashed lines - one narrower and the other closer to a solid line. A dashed line close to the solid line indicates 2SD (>95% of reference molecules in this region), while a dashed line even closer to the solid line indicates 3SD (>99.7% of reference molecules in this region).

Ribbon diagram models of NKG2D-binding Fab arms in three different orientations (upper panel) and their corresponding surface charge distributions in the same orientation (lower panel) are shown. Three orientations are shown: both frontal (left panel: front view; middle panel: back view) and antigen-binding surface (right panel: top view).

CDR length and surface hydrophobicity analysis patches of the NKG2D targeting arm of AB0192 are shown. Arrows indicate where the NKG2D-targeting Fab of AB0192 is positioned relative to advanced clinical-stage mAbs. The inner dashed line indicates the 2SD (>95% of reference molecules within this region). The outermost dotted line indicates the 3SD (>99.7% of reference molecules within this region).

Analysis of patches containing positive and negative charges and the symmetry of these charges around the CDR regions is shown. Arrows indicate the location of the NKG2D-targeting Fab of AB0192 with reference to a database of 377 late-stage therapeutic antibodies. Each plot has two dashed lines - one narrower and the other closer to a solid line. A dashed line closer to the solid line indicates 2SD (>95% of reference molecules in this region), while a dashed line closer to the solid line indicates 3SD (>99.7% of reference molecules in this region). Analysis of patches containing positive and negative charges and the symmetry of these charges around the CDR regions is shown. Arrows indicate the location of the NKG2D-targeting Fab of AB0192 with reference to a database of 377 late-stage therapeutic antibodies. Each plot has two dashed lines - one narrower and the other closer to a solid line. A dashed line closer to the solid line indicates 2SD (>95% of reference molecules in this region), while a dashed line closer to the solid line indicates 3SD (>99.7% of reference molecules in this region).

A and B show the position of the AB0192 chain on the Treg-adjusted EpiMatrix protein immunogenicity scale. A shows a cartoon representation of AB0192. B shows the position of chains H, S, and L of AB0192 on the Treg-regulated immunogenic protein scale. A and B show the position of the AB0192 chain on the Treg-adjusted EpiMatrix protein immunogenicity scale. A shows a cartoon representation of AB0192. B shows the position of chains H, S, and L of AB0192 on the Treg-regulated immunogenic protein scale.

Figure 3 shows a schematic of the two-step platform purification of AB0192. Left panel: SDS-PAGE analysis of AB0192 before and after the first step of the purification process (protein A capture). Middle panel: SEC analysis of AB0192 after protein A step and after cIEX step. Right panel: reducing and non-reducing SDS-PAGE of purified AB0192. Figure 3 shows a schematic of the two-step platform purification of AB0192. Left panel: SDS-PAGE analysis of AB0192 before and after the first step of the purification process (protein A capture). Middle panel: SEC analysis of AB0192 after protein A step and after cIEX step. Right panel: reducing and non-reducing SDS-PAGE of purified AB0192. Figure 3 shows a schematic of the two-step platform purification of AB0192. Left panel: SDS-PAGE analysis of AB0192 before and after the first step of the purification process (protein A capture). Middle panel: SEC analysis of AB0192 after protein A step and after cIEX step. Right panel: reducing and non-reducing SDS-PAGE of purified AB0192. Figure 3 shows a schematic of the two-step platform purification of AB0192. Left panel: SDS-PAGE analysis of AB0192 before and after the first step of the purification process (protein A capture). Middle panel: SEC analysis of AB0192 after protein A step and after cIEX step. Right panel: reducing and non-reducing SDS-PAGE of purified AB0192.

Figure 3 shows intact mass analysis of AB0192 (Lot AB0192-005) using LC-MS analysis. AB0192 theoretical mass = 126,429.5 Da. AB0192 lot AB0192-005 observed mass = 126, 429.9 Da.

Shown is SEC (Superdex 200) analysis of purified AB0192 (lot AB0192-005), which showed greater than 99% monomer as determined by integrated peak area.

Purity of AB0192 (lot AB0192-005) under non-reducing (“NR”; left panel) and reducing (“R”; right panel) conditions by CE-SDS. Purity of AB0192 (lot AB0192-005) under non-reducing (“NR”; left panel) and reducing (“R”; right panel) conditions by CE-SDS.

Figure 10 shows the cIEF profile of AB0192 (Lot AB0192-005).

Figure 3 shows the hydrophobicity analysis of AB0192 by HIC.

Figures 225A-225C show DSC analysis of AB0192 in three different buffer systems. PBS pH 7.4 (A), HST, pH 6.0 (B) and CST, pH 7.0 (C). Figures 225A-225C show DSC analysis of AB0192 in three different buffer systems. PBS pH 7.4 (A), HST, pH 6.0 (B) and CST, pH 7.0 (C). Figures 225A-225C show DSC analysis of AB0192 in three different buffer systems. PBS pH 7.4 (A), HST, pH 6.0 (B) and CST, pH 7.0 (C).

Figure 3 shows the predicted disulfide map of AB0192.

Extracted ion chromatograms (XIC) of engineered disulfide pairs of scFv (non-reduced and reduced) and the strongest charge states of the peptide pairs are shown.

Shows the presence of a CH3-CH3 intermolecular Fc engineered disulfide.

Binding of AB0192 to human CLEC12A tested by SPR. Binding of AB0192 to human CLEC12A tested by SPR.

Figure 3 shows binding of AB0192 to genetic variants of human CLEC12A (Q244) compared to CLEC12A (K244) WT and cynomolgus monkey cyno CLEC12A tested by SPR. Figure 3 shows binding of AB0192 to genetic variants of human CLEC12A (Q244) compared to CLEC12A (K244) WT and cynomolgus monkey cyno CLEC12A tested by SPR.

Binding of AB0192 to differentially glycosylated CLEC12A tested by SPR. Binding of AB0192 to differentially glycosylated CLEC12A tested by SPR.

Binding of AB0192 to CLEC12A+ isogenic and to HL60 and PL21 AML cells.

Figure 3 shows binding of AB0192 to human NKG2D tested by SPR. Figure 3 shows binding of AB0192 to human NKG2D tested by SPR. Figure 3 shows binding of AB0192 to human NKG2D tested by SPR.

Figure 3 shows binding of AB0192 to the hCD16a V158 allele. Figure 3 shows binding of AB0192 to the hCD16a V158 allele. Figure 3 shows binding of AB0192 to the hCD16a V158 allele.

AB0192 binds CD16a and NKG2D simultaneously, resulting in strong binding. AB0192 was induced by NKG2D (middle line shown on the right side of the graph), CD16a (bottom line shown on the right side of the graph), or mixed CD16a and NKG2D (top line shown on the right side of the graph). lines). Note that the surface has not been optimized for maximum affinity efficiency.

Figure 2 shows that captured AB0192 is sequentially saturated with hCLEC12A and hNKG2D. Targets were injected sequentially over AB0192 captured on a Biacore chip. Top panel represents binding of hCLEC12A (100 nM) followed by binding of hNKG2D (7 μM) to captured AB0192. The bottom panel shows the reverse order of target binding (human NKG2D binding followed by hCLEC12A). The relative binding stoichiometry (compared to the binding of each target to unoccupied captured AB0192) is shown in Table 218. Figure 2 shows that captured AB0192 is sequentially saturated with hCLEC12A and hNKG2D. Targets were injected sequentially over AB0192 captured on a Biacore chip. Top panel represents binding of hCLEC12A (100 nM) followed by binding of hNKG2D (7 μM) to captured AB0192. The bottom panel shows the reverse order of target binding (human NKG2D binding followed by hCLEC12A). The relative binding stoichiometry (compared to the binding of each target to unoccupied captured AB0192) is shown in Table 218.

Binding of AB0192 and trastuzumab to recombinant human CD64 (FcγRI). Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD64 (FcγRI). Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD64 (FcγRI). Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD64 (FcγRI). Differences in apparent maximal binding responses between molecules are due to differences in molecular weight.

Binding of AB0192 and trastuzumab to recombinant human CD64 (FcγRI). Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD64 (FcγRI). Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD64 (FcγRI). Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD64 (FcγRI). Differences in apparent maximal binding responses between molecules are due to differences in molecular weight.

Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) H131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) H131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) H131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) H131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) H131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) H131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) H131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) H131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight.

Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) R131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) R131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) R131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) R131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) R131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) R131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) R131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32a (FcγRIIa) R131 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight.

Binding of AB0192 and trastuzumab to recombinant human CD16a (FcγRIIIa) F158 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD16a (FcγRIIIa) F158 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD16a (FcγRIIIa) F158 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD16a (FcγRIIIa) F158 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD16a (FcγRIIIa) F158 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD16a (FcγRIIIa) F158 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD16a (FcγRIIIa) F158 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD16a (FcγRIIIa) F158 alleles. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight.

Binding of AB0192 and trastuzumab to recombinant cynomolgus monkey CD16 (FcγRIII). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant cynomolgus monkey CD16 (FcγRIII). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant cynomolgus monkey CD16 (FcγRIII). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant cynomolgus monkey CD16 (FcγRIII). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight.

Binding of AB0192 and trastuzumab to recombinant human CD32b (FcγRIIb). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32b (FcγRIIb). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32b (FcγRIIb). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32b (FcγRIIb). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32b (FcγRIIb). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32b (FcγRIIb). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32b (FcγRIIb). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD32b (FcγRIIb). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight.

Binding of AB0192 and trastuzumab to recombinant human CD16b (FcγRIIIb). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD16b (FcγRIIIb). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD16b (FcγRIIIb). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human CD16b (FcγRIIIb). The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight.

Binding of AB0192 and trastuzumab to recombinant human FcRn. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human FcRn. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human FcRn. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human FcRn. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human FcRn. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human FcRn. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human FcRn. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant human FcRn. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight.

Binding of AB0192 and trastuzumab to recombinant cynomolgus monkey FcRn. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant cynomolgus monkey FcRn. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant cynomolgus monkey FcRn. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight. Binding of AB0192 and trastuzumab to recombinant cynomolgus monkey FcRn. The top panel of each molecule represents the raw binding sensorgram, while the bottom panel represents the fit to the steady-state affinity model. Differences in apparent maximal binding responses between molecules are due to differences in molecular weight.

Epitope binning of AB0192 against other CLEC12A-binding TriNKETs.

Shows the potency of AB0192 in the primary NK-mediated cytotoxicity assay compared to the corresponding mAb control.

A and B show non-specific analysis of AB0192 in PSR assay. A: Schematic of the PSR assay; B: PSR binding AB0089 compared to an antibody with known PSR binding (rituximab) and an antibody with no known non-specificity (trastuzumab). A and B show non-specific analysis of AB0192 in PSR assay. A: Schematic of the PSR assay; B: PSR binding AB0089 compared to an antibody with known PSR binding (rituximab) and an antibody with no known non-specificity (trastuzumab). A and B show non-specific analysis of AB0192 in PSR assay. A: Schematic of the PSR assay; B: PSR binding AB0089 compared to an antibody with known PSR binding (rituximab) and an antibody with no known non-specificity (trastuzumab).

Figure 10 shows the relative Z-scores of AB0089 in the HuProt™ Human Proteome Microarray Assay. Plot showing relative binding Z-scores (y-axis) versus the top 24 identified binding partners of AB0089 (x-axis). hCLEC12a is plotted at position 1 with a Z-score of 150.06.

Figure 3 shows SEC analysis of AB0192 after 4 weeks at pH 6.0 at 40°C in HST compared to control. Figure 3 shows SEC analysis of AB0192 after 4 weeks at pH 6.0 at 40°C in HST compared to control.

Shows CE-SDS analysis of AB0192 after 4 weeks at pH 6.0 at 40° C. in HST compared to control. Non-reduced conditions are displayed in the left panel. The reduced state is displayed in the right panel. Shows CE-SDS analysis of AB0192 after 4 weeks at pH 6.0 at 40° C. in HST compared to control. Non-reduced conditions are displayed in the left panel. The reduced state is displayed in the right panel.

AB0192 is stable in HST at pH 6.0 and after stress at 40° C. for 4 weeks: indicating no effect on CLEC12A, NKG2D, or CD16a binding. AB0192 is stable in HST at pH 6.0 and after stress at 40° C. for 4 weeks: indicating no effect on CLEC12A, NKG2D, or CD16a binding. AB0192 is stable in HST at pH 6.0 and after stress at 40° C. for 4 weeks: indicating no effect on CLEC12A, NKG2D, or CD16a binding.

FIG. 10 shows that there was no difference in potency between control samples and stressed samples of AB0192 after 4 weeks at pH 6.0 at 40° C. in the KHYG-1-CD16a-mediated cytotoxicity assay in HST. .

It shows minimal aggregation of AB0192 in CST after 4 weeks at pH 7.0 at 40° C. as determined by SEC analysis. It shows minimal aggregation of AB0192 in CST after 4 weeks at pH 7.0 at 40° C. as determined by SEC analysis.

It shows minimal fragmentation of AB0192 after 4 weeks at 40° C. at pH 7.0 in CST compared to controls, as determined by CE-SDS analysis. It shows minimal fragmentation of AB0192 after 4 weeks at 40° C. at pH 7.0 in CST compared to controls, as determined by CE-SDS analysis.

AB0192 is stable in CST at pH 7.0 and after stress at 40° C. for 4 weeks: showing no effect on CLEC12A, NKG2D, or CD16a binding. AB0192 is stable in CST at pH 7.0 and after stress at 40° C. for 4 weeks: showing no effect on CLEC12A, NKG2D, or CD16a binding. AB0192 is stable in CST at pH 7.0 and after stress at 40° C. for 4 weeks: showing no effect on CLEC12A, NKG2D, or CD16a binding.

It shows that there was no difference in potency between control samples and stressed samples of AB0192 after 4 weeks at pH 7.0 at 40° C. in the KHYG-1-CD16a cytotoxicity assay in CST.

SEC analysis of AB0192 after forced oxidative stress compared to control shows no significant decrease in monomer content. SEC analysis of AB0192 after forced oxidative stress compared to control shows no significant decrease in monomer content.

Shows CE-SDS analysis of AB0192 after forced oxidation compared to control, showing that purity remains high. Shows CE-SDS analysis of AB0192 after forced oxidation compared to control, showing that purity remains high.

AB0192 is stable after oxidative stress (pH 5.0, 40° C., 14 days); shows no effect on CLEC12A, NKG2D, or CD16a binding. Small differences in maximal binding signals reflect differences in capture levels. AB0192 is stable after oxidative stress (pH 5.0, 40° C., 14 days); shows no effect on CLEC12A, NKG2D, or CD16a binding. Small differences in maximal binding signals reflect differences in capture levels. AB0192 is stable after oxidative stress (pH 5.0, 40° C., 14 days); shows no effect on CLEC12A, NKG2D, or CD16a binding. Small differences in maximal binding signals reflect differences in capture levels.

Figure 2 shows that the potency of AB0192 after forced oxidation in the KHYG-1-CD16a mediated cytotoxicity assay was similar to control samples.

SEC analysis of AB0192 after prolonged low pH exposure (pH 5.0, 40° C., 14 days) shows no loss of monomer. SEC analysis of AB0192 after prolonged low pH exposure (pH 5.0, 40° C., 14 days) shows no loss of monomer.

Shows CE-SDS analysis of AB0192 after prolonged low pH stress (pH 5.0, 40° C., 14 days) with less than 1% difference in purity detected by non-reduced and reduced CE-SDS. was shown. Non-reduced conditions are displayed in the left panel. The reduced state is displayed in the right panel. Shows CE-SDS analysis of AB0192 after prolonged low pH stress (pH 5.0, 40° C., 14 days) with less than 1% difference in purity detected by non-reduced and reduced CE-SDS. was shown. Non-reduced conditions are displayed in the left panel. The reduced state is displayed in the right panel.

AB0192 is stable after low pH stress (pH 5.0, 40° C., 14 days); binding of CLEC12A, NKG2D, or CD16a was not affected. AB0192 is stable after low pH stress (pH 5.0, 40° C., 14 days); binding of CLEC12A, NKG2D, or CD16a was not affected. AB0192 is stable after low pH stress (pH 5.0, 40° C., 14 days); binding of CLEC12A, NKG2D, or CD16a was not affected.

Figure 3 shows charge analysis of AB0192 after prolonged low pH stress (pH 5.0, 40°C, 14 days) by cIEF, showing no difference between control and pH 5 stressed samples.

We show that the potency of AB0192 after prolonged pH 5.0 stress was unchanged compared to controls in the KHYG-1-CD16a cytotoxicity assay.

SEC analysis of AB0192 after prolonged high pH stress (pH 8.0, 40° C., 14 days) shows that there was 1.6% loss of monomer. SEC analysis of AB0192 after prolonged high pH stress (pH 8.0, 40° C., 14 days) shows that there was 1.6% loss of monomer.

CE-SDS analysis of AB0192 after prolonged high pH stress (pH 8.0, 40° C., 14 days) is shown. Slight differences in purity were detected under non-reducing (left) and reducing (right) conditions. CE-SDS analysis of AB0192 after prolonged high pH stress (pH 8.0, 40° C., 14 days) is shown. Slight differences in purity were detected under non-reducing (left) and reducing (right) conditions.

AB0192 is stable after high pH stress: no effect on CLEC12A, NKG2D, or CD16a binding. AB0192 is stable after high pH stress: no effect on CLEC12A, NKG2D, or CD16a binding. AB0192 is stable after high pH stress: showing no effect on CLEC12A, NKG2D, or CD16a binding.

Figure 2 shows charge analysis of AB0192 after long-term high pH stress (pH 8.0, 40°C, 14 days) by cIEF, showing an acidic shift in pH 8 stressed samples compared to control samples.

It shows that the potency of AB0192 after long-term exposure to high pH 8.0 stress was not significantly affected as assessed by the KHYG-1-CD16aV cytotoxicity assay.

SEC analysis of AB0192 after 6 cycles of freeze/thaw compared to control showed no loss of monomer. SEC analysis of AB0192 after 6 cycles of freeze/thaw compared to control showed no loss of monomer.

CE-SDS analysis of AB0192 after freeze-thaw stress is shown, showing no loss of purity under either reducing (right panel) or non-reducing (left panel) conditions. CE-SDS analysis of AB0192 after freeze-thaw stress is shown, showing no loss of purity under either reducing (right panel) or non-reducing (left panel) conditions.

SEC analysis of AB0192 after agitation stress compared to control shows no loss of monomer. SEC analysis of AB0192 after agitation stress compared to control shows no loss of monomer.

Shown is the CE-SDS analysis of AB0192 after agitation stress compared to control, showing no increase in fragmentation detected by reduction (right panel) or non-reduction (left panel). Shown is the CE-SDS analysis of AB0192 after agitation stress compared to control, showing no increase in fragmentation detected by reduction (right panel) or non-reduction (left panel).

An SEC analysis of the pH hold sample versus control is shown, which indicated that no change in profile was observed after the low pH hold. An SEC analysis of the pH hold sample versus control is shown, which indicated that no change in profile was observed after the low pH hold.

A cIEF analysis of AB0192 after low pH hold compared to controls is shown, indicating that low pH hold had no measurable effect on the charge profile of AB0192.

AB0192 is stable after low pH holding: no effect on CLEC12A, NKG2D, or CD16a binding. Small differences in maximal binding signals reflect differences in capture levels. AB0192 is stable after low pH holding: no effect on CLEC12A, NKG2D, or CD16a binding. Small differences in maximal binding signals reflect differences in capture levels. AB0192 is stable after low pH holding: no effect on CLEC12A, NKG2D, or CD16a binding. Small differences in maximal binding signals reflect differences in capture levels.

The KHYG1-CD16a mediated cytotoxicity assay showed the potency of AB0192 after the low pH hold, which indicated that the potency of AB0192 was unchanged after the low pH hold step.

AB0089 binding to hCLEC12A before and after low pH holding. Slight differences in absolute maximum signals reflect differences in AB0089 capture levels.

It shows that AB0089 is stable after low pH holding. No difference was observed between the potency of the low pH hold and control samples.

The present application provides multispecific binding proteins that bind to NKG2D and CD16 receptors on natural killer cells and tumor-associated antigens. In some embodiments, the multispecific protein further comprises additional antigen binding sites that bind tumor-associated antigens. The present application also provides pharmaceutical compositions comprising such multispecific binding proteins, and treatments using such multispecific proteins and pharmaceutical compositions for purposes such as treatment of autoimmune diseases and cancer. provide a way. Various aspects of the multispecific binding proteins described in this application are described in the sections below; however, the various aspects of the multispecific binding proteins described in one particular section may be used in any particular It is not limited to sections either.

To facilitate understanding of this application, a number of terms and phrases are defined below.

As used herein, the terms "a" and "an" mean "one or more" and include plural forms unless the context dictates otherwise.

As used herein, the term "antigen-binding site" refers to the portion of the immunoglobulin molecule that participates in antigen binding. In human antibodies, the antigen-binding site is formed by the amino acid residues of the N-terminal variable (“V”) regions of the heavy (“H”) and light (“L”) chains. Three highly divergent stretches within the V regions of the heavy and light chains, called "hypervariable regions," are interposed between more conserved flanking stretches known as "framework regions" or "FRs." be. Thus, the term "FR" refers to amino acid sequences that are naturally found between and adjacent to hypervariable regions in immunoglobulins. In human antibody molecules, the three hypervariable regions of the light chain and the three hypervariable regions of the heavy chain are arranged relative to each other in three-dimensional space to form the antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and the three hypervariable regions of each heavy and light chain are called "complementarity determining regions" or "CDRs." In certain animals, such as camels and cartilaginous fish, the antigen-binding site is formed by a single antibody chain, providing a "single-domain antibody." The antigen-binding site can be an intact antibody, an antigen-binding fragment of an antibody that retains the antigen-binding surface, or a peptide linker to connect the heavy chain variable domain to the light chain variable domain of a single polypeptide. can be present in recombinant polypeptides such as scFvs.

As used herein, the term "tumor-associated antigen" refers to any antigen including, but not limited to, cancer-associated proteins, glycoproteins, gangliosides, carbohydrates, lipids. Such antigens can be expressed on malignant cells or in the tumor microenvironment, such as tumor-associated blood vessels, extracellular matrix, mesenchymal stroma, or immune infiltration. In preferred embodiments of the present disclosure, the term "tumor-associated antigen" refers to CLEC12A.

As used herein, the terms "subject" and "patient" refer to organisms treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., mice, monkeys, horses, cows, pigs, dogs, cats, etc.), and more preferably humans. mentioned.

As used herein, the term "effective amount" refers to a sufficient amount of a compound (eg, a compound of the invention) to effect beneficial or desired results. An effective amount may be administered in one or more administrations, applications or dosages and is not intended to be limited to any particular formulation or route of administration. As used herein, the term "treating" means any effect, e.g. Including improvement of symptoms.

As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and an inert or active carrier that renders the composition inherently suitable for in vivo or ex vivo therapeutic use. Point to combination.

As used herein, the term "pharmaceutically acceptable carrier" refers to any standard pharmaceutical carrier such as phosphate buffered saline, water, emulsions (e.g. oil-in-water or water-in-oil emulsions), and various types of wetting agents. The composition may also contain stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, for example, Martin, Remington's Pharmaceutical Sciences, 15th Ed. , Mack Publ. Co. , Easton, PA [1975].

As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound described in this application that is refers to a salt that is capable of providing a compound described in this application or an active metabolite or residue thereof after administration of . "Salts" of the compounds described in this application can be derived from inorganic or organic acids and bases, as is known to those skilled in the art. Exemplary acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene- p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, ethanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid and the like. Other acids, such as oxalic acid, are not pharmaceutically acceptable per se, but are useful as intermediates in obtaining the compounds described in this application and their pharmaceutically acceptable acid addition salts. It can be employed in the preparation of salts.

Examples of bases include, but are not limited to, alkali metal (e.g., sodium) hydroxides, alkaline earth metal (e.g., magnesium) hydroxides, ammonia, and formula NW ₄ ⁺ where W is , C _1-4 alkyl) and the like.

Exemplary salts include, but are not limited to, acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, hydrogen sulfate, butyrate, citrate, camphorate. salt, camphorsulphonate, cyclopentanepropionate, digluconate, dodecylsulphate, ethanesulphonate, fumarate, flucoheptanoate, glycerophosphate, hemisulphate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, Palmate, pectate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, etc. . Other examples of salts are described in this application combined with suitable cations such as Na ⁺ , NH ₄ ⁺ , and NW ₄ ⁺ where W is a C _1-4 alkyl group. and the anion of the compound.

For therapeutic use, salts of the compounds described in this application are considered pharmaceutically acceptable. However, salts of acids and bases that are non-pharmaceutically acceptable may find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

As used herein, CLEC12A (also known as CLL-1, DCAL-2, MICL, and CD371) refers to the protein of Uniprot accession number Q5QGZ9 and related isoforms and orthologs.

Throughout the description, when compositions are described as having, contain, or include particular components, or when processes and methods are described as having, include, or include particular steps, enumeration There are compositions described in the present application that consist essentially of or consist of the components listed, and there are processes and methods according to the present application that consist essentially of or consist of the recited processing steps It is further contemplated to do.

As a general matter, compositions specifying percentages are by weight unless otherwise specified. Furthermore, if a variable is not accompanied by a definition, the previous definition of that variable takes precedence.

I. Proteins The present application provides multispecific binding proteins that bind to the NKG2D and CD16 receptors on natural killer cells and CLEC12A. Multispecific binding proteins are useful in the pharmaceutical compositions and therapeutic methods described herein. Binding of the multispecific binding protein to the NKG2D and CD16 receptors on natural killer cells enhances the activity of natural killer cells towards destruction of tumor cell expressing tumor cells. Binding of the multispecific binding protein to tumor cells expressing tumor antigens brings these cells into close proximity to natural killer cells and facilitates direct and indirect destruction of tumor cells by natural killer cells. Multispecific binding proteins that bind NKG2D, CD16, and other targets are disclosed in International Application Publication Nos. WO2018148445 and WO2019157366 (not incorporated herein by reference). Further description of some exemplary multispecific binding proteins is provided below.

The first component of the multispecific binding protein is an antigen-binding site that binds to NKG2D receptor-expressing cells, including but not limited to NK cells, γδ T cells, and CD8 ⁺ αβ T cells. mentioned. Upon NKG2D binding, multispecific binding proteins can block natural ligands such as ULBP6 and MICA from binding to NKG2D and activating NK cells.

The second component of the multispecific binding protein is the antigen-binding site that binds the tumor-associated antigen, CLEC12A. Tumor-associated antigen-expressing cells include, for example, gastric cancer, esophageal cancer, lung cancer, breast cancer (e.g., triple-negative breast cancer), colorectal cancer, ovarian cancer, esophageal cancer, uterine cancer, cervical cancer, head and neck cancer, glioma, and in solid tumor indications such as pancreatic cancer, or certain hematological malignancies such as acute myeloid leukemia.

A third component of the multispecific binding protein expresses CD16, an Fc receptor on the surface of leukocytes including natural killer cells, macrophages, neutrophils, eosinophils, mast cells and follicular dendritic cells. An antibody Fc domain or portion thereof or antigen binding site that binds to cells.

Additional antigen binding sites of the multispecific binding protein may bind the same tumor-associated antigen. In certain embodiments, the first antigen binding site that binds NKG2D is a scFv and the second and additional antigen binding sites that bind tumor-associated antigens are each Fab fragments. In certain embodiments, the first antigen binding site that binds NKG2D is a scFv and the second and additional antigen binding sites that bind tumor-associated antigen are each scFv. In certain embodiments, the first antigen binding site that binds NKG2D is a Fab fragment and the second and additional antigen binding sites that bind tumor-associated antigen are each scFv. In certain embodiments, the first antigen binding site that binds NKG2D is Fab and the second and additional antigen binding sites that bind tumor-associated antigen are each Fab fragments.

Each of the antigen binding sites may incorporate an antibody heavy chain variable domain and an antibody light chain variable domain (e.g., arranged like an antibody or fused together to form a scFv), or one or more The antigen-binding site of may be a single domain antibody, such as a _VHH antibody, such as the camel antibody, or a _VNAR antibody, such as the antibody found in cartilaginous fish.

In some embodiments, the second antigen binding site incorporates a light chain variable domain having an amino acid sequence identical to the amino acid sequence of the light chain variable domain present in the first antigen binding site.

Multispecific binding proteins described herein can take a variety of formats. For example, one format is a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a first immunoglobulin light chain, a second immunoglobulin heavy chain and a second immunoglobulin light chain. Yes (Fig. 1). This first immunoglobulin heavy chain comprises a first Fc (hinge-CH2-CH3) domain, a first heavy chain variable domain, and optionally a first CH1 heavy chain domain. This first immunoglobulin light chain comprises a first light chain variable domain and optionally a first light chain constant domain. This first immunoglobulin light chain, together with the first immunoglobulin heavy chain, forms an antigen binding site that binds NKG2D. This second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain, and optionally a second CH1 heavy chain domain. This second immunoglobulin light chain comprises a second light chain variable domain and optionally a second light chain constant domain. This second immunoglobulin light chain, together with the second immunoglobulin heavy chain, forms an antigen binding site that binds CLEC12A. In some embodiments, the first Fc domain and the second Fc domain together are capable of binding CD16 (Figure 1). In some embodiments, this first immunoglobulin light chain is identical to the second immunoglobulin light chain.

Another exemplary format includes a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain, a second immunoglobulin heavy chain, and an immunoglobulin light chain (eg, FIG. 2A). In some embodiments, the first immunoglobulin heavy chain is paired with a heavy and light chain variable domain (NKG2D or CLEC12A), either via a linker or antibody hinge. It contains a first Fc (hinge-CH2-CH3) domain fused to a single-chain variable fragment (scFv) composed of the binding). This second immunoglobulin heavy chain comprises a second Fc (hinge-CH2-CH3) domain, a second heavy chain variable domain and a CH1 heavy chain domain. This immunoglobulin light chain comprises a light chain variable domain and a light chain constant domain. In some embodiments, the second immunoglobulin heavy chain pairs with the immunoglobulin light chain and binds NKG2D or binds a tumor-associated antigen, wherein the first Fc domain binds to NKG2D The second immunoglobulin heavy chain paired with the immunoglobulin light chain when fused to the binding scFv binds to the tumor-associated antigen, but not to NKG2D, and vice versa. is. In some embodiments, the scFv in the first immunoglobulin heavy chain binding binds CLEC12A; and the heavy chain variable domain of the second immunoglobulin heavy chain and the light chain variable domain of the immunoglobulin light chain bind to , it binds to NKG2D (eg, FIG. 2E). In some embodiments, the scFv of the first immunoglobulin heavy chain binds NKG2D; and the heavy chain variable domain of the second immunoglobulin heavy chain and the light chain variable domain of the immunoglobulin light chain bind to , it binds to CLEC12A. In some embodiments, the first Fc domain and the second Fc domain together can bind CD16 (eg, FIG. 2A).

Another exemplary format includes a heterodimeric multispecific antibody comprising a first immunoglobulin heavy chain and a second immunoglobulin heavy chain (eg, FIG. 2B). In some embodiments, the first immunoglobulin heavy chain is paired with a heavy and light chain variable domain (NKG2D or CLEC12A), either via a linker or antibody hinge. It contains a first Fc (hinge-CH2-CH3) domain fused to a single-chain variable fragment (scFv) composed of the binding). In some embodiments, the second immunoglobulin heavy chain is paired with a heavy and light variable domain (NKG2D or tumor-associated a second Fc (hinge-CH2-CH3) domain fused to a single-chain variable fragment (scFv) composed of a single chain variable fragment (scFv) that binds antigen, where the first Fc domain is fused to the scFv that binds NKG2D The proviso is that the second Fc domain fused to the scFv then binds the tumor-associated antigen, but not NKG2D, and vice versa. In some embodiments, the first Fc domain and the second Fc domain together can bind CD16 (eg, FIG. 2B).

In some embodiments, the single chain variable fragment (scFv) described above is linked to the antibody constant domain via a hinge sequence. In some embodiments, the hinge comprises amino acids Ala-Ser or Gly-Ser. In some embodiments, the hinge connects the scFv that bind NKG2D and the antibody heavy chain constant domain comprises amino acids Ala-Ser. In some embodiments, the hinge connects scFvs that bind tumor-associated antigens and the antibody heavy chain constant domain comprises the amino acids Gly-Ser. In some other embodiments, the hinge comprises amino acids Ala-Ser and Thr-Lys-Gly. The hinge sequence provides flexibility of binding to the target antigen and can be a balance between flexibility and optimal shape.

In some embodiments, the single chain variable fragment (scFv) described above comprises a heavy chain variable domain and a light chain variable domain. In some embodiments, the heavy chain variable domain forms a disulfide bridge with the light chain variable domain to enhance scFv stability. For example, a disulfide bridge can be formed between residue C44 of the heavy chain variable domain and C100 residue of the light chain variable domain (amino acid positions are numbered under Kabat). In some embodiments, the heavy chain variable domain is linked to the light chain variable domain via a flexible linker. Any suitable linker can be used, such as the (G ₄ S) ₄ linker ((GlyGlyGlyGlySer) ₄ (SEQ ID NO: 119)). In some scFv embodiments, the heavy chain variable domain is located N-terminal to the light chain variable domain. In some scFv embodiments, the heavy chain variable domain is located C-terminal to the light chain variable domain.

The multispecific binding proteins described herein may further comprise one or more additional antigen binding sites. Additional antigen binding site(s) may be fused to the N-terminus of the constant region CH2 domain or the C-terminus of the constant region CH3 domain, optionally via a linker sequence. In certain embodiments, the additional antigen binding site(s) is in the form of a single chain variable region (scFv), optionally disulfide stabilized, resulting in a tetravalent or trivalent multispecific binding protein. take. For example, a multispecific binding protein may comprise a first antigen binding site that binds NKG2D, a second antigen binding site that binds CLEC12A, an additional antigen binding site that binds a tumor-associated antigen, and an antibody constant region or portion thereof. portion (sufficient to bind to CD16 or a fourth antigen binding site that binds to CD16). Any of these antigen binding sites may take the form of either Fab fragments or scFvs such as the scFvs described above.

In some embodiments, the additional antigen binding site binds to a different epitope of the tumor-associated antigen than the second antigen binding site. In some embodiments, the additional antigen binding site binds to the same epitope as the second antigen binding site. In some embodiments, the additional antigen binding site comprises the same heavy and light chain CDR sequences as the second antigen binding site. In some embodiments, the additional antigen binding site comprises the same heavy and light chain variable domain sequences as the second antigen binding site. In some embodiments, the additional antigen binding site has the same amino acid sequence(s) as the second antigen binding site. In some embodiments, the additional antigen binding site comprises heavy and light chain variable domain sequences that differ from the heavy and light chain variable domain sequences of the second antigen binding site. In some embodiments, the additional antigen binding site has an amino acid sequence that differs from the sequence of the second antigen binding site. In some embodiments, the second antigen binding site and the additional antigen binding site bind different tumor-associated antigens. In some embodiments, the second antigen binding site and the additional antigen binding site bind different antigens. An exemplary format is shown in Figures 2C and 2D. Thus, multispecific binding proteins can provide bivalent engagement of tumor-associated antigens. Bivalent engagement of tumor-associated antigens by multispecific proteins can stabilize tumor-associated antigens on the surface of tumor cells and enhance NK cell cytotoxicity against tumor cells. Bivalent engagement of tumor-associated antigens by multispecific proteins may confer stronger binding of the multispecific proteins to tumor cells, thereby targeting tumor cells, especially low levels of tumor-associated antigens. Promotes a stronger cytotoxic response of NK cells towards the expressing tumor cells.

Multispecific binding proteins can take additional formats. In some embodiments, the multispecific binding protein is the triomab form, a trifunctional bispecific antibody that maintains an IgG-like shape. This chimera consists of two half-antibodies, each with one light chain and one heavy chain, derived from the two parental antibodies.

In some embodiments, the multispecific binding protein is in KiH form, which includes knob-into-hole (KiH) technology. KiH involves engineering the C _H 3 domain to create “knobs” or “holes” in each heavy chain to facilitate heterodimerization. The concept behind the "Knobs-into-Holes (KiH)" Fc technology is to replace small residues with bulky residues (e.g., T366W _CH3A with EU numbering) to construct a single CH3 domain. The goal was to introduce a "knob" into (CH3A). Complementary "hole" surface on the other CH3 domain (CH3B) by replacing the closest flanking residue to the knob with a smaller residue (e.g. T366S/L368A/Y407V _CH3B ) to accommodate the "knob" It was created. "Whole" mutations are identified by structured guided phage library screening (Atwell S, Ridgway JB, Wells JA, Carter P., Stable heterodimers from remodeling the domain interface of a homodimer using a phage, Jol. 1997) 270(1):26-35). ＫｉＨ ＦｃバリアントのＸ線結晶構造（Ｅｌｌｉｏｔｔ ＪＭ、Ｕｌｔｓｃｈ Ｍ、Ｌｅｅ Ｊ、Ｔｏｎｇ Ｒ、Ｔａｋｅｄａ Ｋ、Ｓｐｉｅｓｓ Ｃ、ｅｔ ａｌ．、Ａｎｔｉｐａｒａｌｌｅｌ ｃｏｎｆｏｒｍａｔｉｏｎ ｏｆ ｋｎｏｂ ａｎｄ ｈｏｌｅ ａｇｌｙｃｏｓｙｌａｔｅｄ ｈａｌｆ－ａｎｔｉｂｏｄｙ ｈｏｍｏｄｉｍｅｒｓ ｉｓ ｍｅｄｉａｔｅｄ ｂｙ ａ ＣＨ２－ＣＨ３ ｈｙｄｒｏｐｈｏｂｉｃ ｉｎｔｅｒａｃｔｉｏｎ．Ｊ．Ｍｏｌ．Ｂｉｏｌ．（２０１４）４２６（９）：１９４７－５７；Ｍｉｍｏｔｏ Ｆ、Ｋａｄｏｎｏ Ｓ、Ｋａｔａｄａ Ｈ、Ｉｇａｗａ Ｔ、Ｋａｍｉｋａｗａ Ｔ、Ｈａｔｔｏｒｉ Ｋ．Ｃｒｙｓｔａｌ ｓｔｒｕｃｔｕｒｅ ｏｆ ａ ｎｏｖｅｌ ａｓｙｍｍｅｔｒｉｃａｌｌｙ ｅｎｇｉｎｅｅｒｅｄ Ｆｃ ｖａｒｉａｎｔ ｗｉｔｈ ｉｍｐｒｏｖｅｄ ａｆｆｉｎｉｔｙ for FcγRs.Mol.Immunol.(2014) 58(1):132-8), heterodimerization is thermodynamically driven by hydrophobic interactions driven by steric complementarity at the core interface between CH3 domains. , whereas knob-knob and hole-hole interfaces were demonstrated not to favor homodimerization due to steric hindrance and disruption of favorable interactions, respectively.

In some embodiments, the multispecific binding protein is a dual variable domain immunoglobulin (DVD-Ig™) form, which connects two monoclonal antibodies via a flexible naturally occurring linker. Combining the target binding domains results in a tetravalent IgG-like molecule.

In some embodiments, the multispecific binding proteins are in orthogonal Fab interface (ortho-Fab) format.オルトＦａｂ ＩｇＧアプローチ（Ｌｅｗｉｓ ＳＭ、Ｗｕ Ｘ、Ｐｕｓｔｉｌｎｉｋ Ａ、Ｓｅｒｅｎｏ Ａ、Ｈｕａｎｇ Ｆ、Ｒｉｃｋ ＨＬ、ｅｔ ａｌ．、Ｇｅｎｅｒａｔｉｏｎ ｏｆ ｂｉｓｐｅｃｉｆｉｃ ＩｇＧ ａｎｔｉｂｏｄｉｅｓ ｂｙ ｓｔｒｕｃｔｕｒｅ－ｂａｓｅｄ ｄｅｓｉｇｎ ｏｆ ａｎ ｏｒｔｈｏｇｏｎａｌ Ｆａｂ ｉｎｔｅｒｆａｃｅ．Ｎａｔ．Ｂｉｏｔｅｃｈｎｏｌ．（２０１４） 32(2):191-8), structure-based region design introduced complementary mutations to the LC and HC _VH-CH1 interfaces in only one Fab fragment without altering the other Fab fragments. be.

In some embodiments, the multispecific binding protein is a 2-in-1 Ig format. In some embodiments, the multispecific binding protein is in the ES form, which is a heterodimeric construct comprising two different Fab fragments that bind Target 1 and Target 2 fused to Fc. Heterodimerization is ensured by electrostatic steering mutations of Fc.

In some embodiments, the multispecific binding protein is a κλ-Body form, which is a heterodimeric construct having two different Fab fragments fused to an Fc stabilized by heterodimerization mutations : Fab fragment 1 targeting antigen 1 contains kappa LC and Fab fragment 2 targeting antigen 2 contains lambda LC. FIG. 13A is an exemplary representation of one form of κλ-Body. FIG. 13B is an exemplary representation of another κλ-Body.

In some embodiments, the multispecific binding protein is in the Fab arm exchange form (by exchanging the heavy chain and attached light chain (half molecule) with a heavy-light chain pair from another molecule, antibodies that exchange Fab fragment arms, resulting in bispecific antibodies).

In some embodiments, the multispecific binding protein is in SEED Body form. The Strand Exchange Engineered Domain (SEED) platform is designed to generate asymmetric, bispecific antibody-like molecules (the ability to extend the therapeutic application of natural antibody therapies). This protein engineering platform is based on the exchange of immunoglobulin structurally related sequences within the conserved CH3 domain. The SEED design allows efficient generation of AG/GA heterodimers, while disfavoring homodimerization of the AG and GA SEED CH3 domains. (Muda M. et al., Protein Eng. Des. Sel. (2011, 24(5):447-54)).

In some embodiments, the multispecific binding protein is in the LuZ-Y form and uses a leucine zipper to induce heterodimerization of two different HCs. (Wranik, BJ. et al., J. Biol. Chem. (2012), 287:43331-9).

In some embodiments, the multispecific binding protein is in Cov-X-Body form. In a bispecific CovX-Body, two different peptides are attached using a branched azetidinone linker and fused site-specifically to a scaffold antibody under mild conditions. The pharmacophore is responsible for functional activity, whereas the antibody scaffold confers a long half-life and Ig-like distribution. Pharmacophores can be chemically optimized or replaced with other pharmacophores to generate optimized or unique bispecific antibodies. (Doppalapudi VR et al., PNAS (2010), 107(52); 22611-22616).

In some embodiments, the multispecific binding protein is an Oasc-Fab heterodimeric form comprising a Fab fragment that binds Target 1 and an scFab that binds Target 2 fused to Fc. Heterodimerization is ensured by Fc mutations.

In some embodiments, the multispecific binding protein is a DuetMab form, which has two different Fab fragments that bind antigens 1 and 2, and an Fc stabilized by heterodimerization mutations. A heterodimeric construct comprising: Fab fragments 1 and 2 contain differential S—S bridges that ensure correct pairing of LC and HC.

In some embodiments, the multispecific binding protein is a cross-mAb form, which consists of two different Fabs binding targets 1 and 2 fused to an Fc stabilized by heterodimerization A heterodimeric construct with a fragment. The CL and CH1 domains and the VH and VL domains are switched, eg, CH1 is fused in-frame with VL and CL is fused in-frame with VH.

In some embodiments, the multispecific binding protein is Fit-Ig, a homodimeric construct in which the Fab fragment that binds Antigen 2 is fused to the N-terminus of the HC of the Fab fragment that binds Antigen 1 form. This construct contains a wild-type Fc.

The individual components of multispecific binding proteins are described in more detail below.

NKG2D-Binding Site Upon binding to the NKG2D and CD16 receptors on natural killer cells, as well as CLEC12A, the multispecific binding protein engages two or more NK-activating receptors, allowing the natural binding to NKG2D. It can block ligand binding. In certain embodiments, the protein is capable of stimulating human NK cells. In some embodiments, the protein can stimulate NK cells in humans and other species such as rodents and cynomolgus monkeys. In some embodiments, the protein can stimulate NK cells in humans as well as other species such as cynomolgus monkeys.

Table 1 lists peptide sequences of heavy and light chain variable domains that in combination can bind to NKG2D. In some embodiments, the heavy and light chain variable domains are arranged in a Fab format. In some embodiments, the heavy and light chain variable domains are fused together to form an scFv.

The NKG2D binding sites listed in Table 1 may differ in their binding affinities to NKG2D, yet they all activate human NK cells.

Unless otherwise indicated, the CDR sequences listed in Table 1 are determined according to Kabat numbering.

In certain embodiments, the first antigen-binding site that binds NKG2D (e.g., human NKG2D) is at least 90% (e.g., at least 91%, at least 92%) the VH of an antibody disclosed in Table 1 , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) an antibody heavy chain variable domain (VH) comprising amino acid sequences that are identical, and at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, for the VL of the same antibody disclosed in Table 1) %, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the first antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 as determined under Kabat (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk A M, (1987), J. Mol. Biol. 196:901-917), MacCallum ( MacCallum RM et al., (1996) J. Mol. Biol. are disclosed in Table 1. In certain embodiments, the first antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of the antibodies disclosed in Table 1.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%) relative to SEQ ID NO:1 , at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), and/or CDR1 of SEQ ID NO: 1 (SEQ ID NO: 2), CDR2 ( SEQ ID NO: 3), and a heavy chain variable domain related to SEQ ID NO: 1, such as by incorporating an amino acid sequence identical to that of CDR3 (SEQ ID NO: 4). A heavy chain variable domain with respect to SEQ ID NO: 1 can combine with various light chain variable domains to form an NKG2D binding site. For example, a first antigen binding site incorporating a heavy chain variable domain related to SEQ ID NO: 1 is , 20, 21, 22, 23, 24, 25, and 46 may further be incorporated. For example, the first antigen binding site is at least 90% relative to SEQ ID NO: 1 (e.g., at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences and SEQ ID NOS: 5, 6, 7, 8, 9, 12, 13, 14, 15, at least 90% (e.g., at least 90%, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%).

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 32, and at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27 or 28, 29, and 30 or 31, respectively (e.g., SEQ ID NOs: 27, 29, and 30, respectively, or SEQ ID NOS: 28, 29, and 31, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 33, 34, and 35, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27 or 28, 29, and 30 or 31, respectively (e.g., SEQ ID NOs: 27, 29, and 30, or SEQ ID NOs: 28, 29, and 31, respectively); and (b) VLs that contain CDR1, CDR2, and CDR3 that comprise the amino acid sequences of SEQ ID NOs: 33, 34, and 35, respectively.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 42, and at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 37 or 38, 39, and 40 or 41, respectively (e.g., SEQ ID NOs: 37, 39, and 40, respectively, or SEQ ID NOS: 38, 39, and 41, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 43, 44, and 45, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 37 or 38, 39, and 40 or 41, respectively (e.g., SEQ ID NOs: 37, 39 and 40, or SEQ ID NOS: 38, 39 and 41, respectively); and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 43, 44, and 45, respectively.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 49, and at least 90% (e.g., at least 91%, VL comprising amino acid sequences that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 27, 29 and 48, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:50, 34, and 51, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 27, 29, and 48, respectively; and (b) SEQ ID NO: 50, respectively; VLs containing CDR1, CDR2, and CDR3 containing 34, and 51 amino acid sequences.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 58, and at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55, and 56 or 57, respectively (e.g., SEQ ID NOs: 53, 55, and 56, respectively, or 54, 55 and 57, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:59, 60, and 61, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55, and 56 or 57, respectively (e.g., SEQ ID NOs: 53, 55 and 56, or SEQ ID NOS: 54, 55 and 57, respectively); and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 59, 60, and 61, respectively.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 68, and at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 63 or 64, 65, and 66 or 67, respectively (e.g., SEQ ID NOs: 63, 65, and 66, respectively, or SEQ ID NOs: 64, 65 and 67, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:59, 60, and 69, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 63 or 64, 55, and 66 or 67, respectively (e.g., SEQ ID NOs: 63, 65 and 66, or SEQ ID NOS: 64, 65 and 67, respectively); and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 59, 60, and 69, respectively.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical, and at least 90% (e.g., at least 91%, VL comprising amino acid sequences that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55, and 90 or 91, respectively (e.g., SEQ ID NOs: 53, 55, and 90, respectively, or 54, 55, and 91, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:93, 44, and 94, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 53 or 54, 55, and 90 or 91, respectively (e.g., SEQ ID NOs: 53, 55 and 90, or SEQ ID NOS: 54, 55 and 91, respectively); and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 93, 44, and 94, respectively.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 75, and at least 90% (e.g., at least 91%, VL comprising amino acid sequences that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 71 or 115, 72, and 73 or 74, respectively (e.g., SEQ ID NOs: 71, 72, and 73, respectively, or SEQ ID NOS: 115, 72, and 74, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:76, 77, and 78, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 71 or 115, 72, and 73 or 74, respectively (e.g., SEQ ID NOs: 71, 72 and 73, or SEQ ID NOS: 115, 72 and 74, respectively); and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 76, 77, and 78, respectively.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 85, and at least 90% (e.g., at least 91%, VL comprising amino acid sequences that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 83 or 84, respectively (e.g., SEQ ID NOs: 80, 82, and 83, respectively, or SEQ ID NOS: 81, 82 and 84, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 83 or 84, respectively (e.g., SEQ ID NOs: 80, 82 and 83, or SEQ ID NOs: 81, 82 and 84, respectively); and (b) VLs containing CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 86, 77, and 87, respectively.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 85, and at least 90% (e.g., at least 91%, VL comprising amino acid sequences that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 96 or 97, respectively (e.g., SEQ ID NOs: 80, 82, and 96, respectively, or SEQ ID NOS: 81, 82, and 97, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 96 or 97, respectively (e.g., SEQ ID NOs: 80, 82 and 96, or SEQ ID NOS: 81, 82 and 97, respectively); and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 86, 77, and 87, respectively.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 85, and at least 90% (e.g., at least 91%, VL comprising amino acid sequences that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 99 or 100, respectively (e.g., SEQ ID NOs: 80, 82, and 99, respectively, or 81, 82 and 100, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 96 or 97, respectively (e.g., SEQ ID NOs: 80, 82 and 99, or SEQ ID NOS: 81, 82 and 100, respectively); and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 86, 77, and 87, respectively.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 85, and at least 90% (e.g., at least 91%, VL comprising amino acid sequences that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 102 or 103, respectively (e.g., SEQ ID NOs: 80, 82, and 102, respectively, or SEQ ID NOs:81, 82 and 103, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 102 or 103, respectively (e.g., SEQ ID NOs: 80, 82 and 102, or SEQ ID NOS: 81, 82 and 103, respectively); and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 86, 77, and 87, respectively.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 85, and at least 90% (e.g., at least 91%, VL comprising amino acid sequences that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 105 or 106, respectively (e.g., SEQ ID NOs: 80, 82, and 105, respectively, or SEQ ID NOs:81, 82 and 106, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 105 or 106, respectively (e.g., SEQ ID NOs: 80, 82 and 105, or SEQ ID NOS: 81, 82 and 106, respectively); and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 86, 77, and 87, respectively.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 85, and at least 90% (e.g., at least 91%, VL comprising amino acid sequences that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 108 or 109, respectively (e.g., SEQ ID NOs: 80, 82, and 108, respectively, or 81, 82 and 109, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 108 or 109, respectively (e.g., SEQ ID NOs: 80, 82 and 108, or SEQ ID NOS:81, 82 and 109, respectively); and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:86, 77, and 87, respectively.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 85, and at least 90% (e.g., at least 91%, VL comprising amino acid sequences that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 111 or 112, respectively (e.g., SEQ ID NOs: 80, 82, and 111, respectively, or 81, 82 and 112, respectively). In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:86, 77, and 87, respectively. In certain embodiments, the first antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 80 or 81, 82, and 111 or 112, respectively (e.g., SEQ ID NOs: 80, 82 and 111, or SEQ ID NOS: 81, 82 and 112, respectively); and (b) a VL comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 86, 77, and 87, respectively.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 114, and at least 90% (e.g., at least 91%, VL comprising amino acid sequences that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical.

In certain embodiments, the first antigen binding site that binds NKG2D is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, A VH comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 117, and at least 90% (e.g., at least 91%, VL comprising amino acid sequences that are at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical.

Multispecific binding proteins can bind NKG2D-expressing cells including, but not limited to, NK cells, γδ T cells and CD8 ⁺ αβ T cells. Upon NKG2D binding, multispecific binding proteins can block natural ligands such as ULBP6 and MICA from binding to NKG2D and activating NK cells.

The multispecific binding protein binds to cells expressing CD16, an Fc receptor on the surface of leukocytes such as natural killer cells, macrophages, neutrophils, eosinophils, mast cells, and follicular dendritic cells. . Proteins of the present disclosure range from 2 nM to 120 nM, such as 20 nM, 2 nM to 10 nM, about 15 nM, about 14 nM, about 13 nM, about 12 nM, about 11 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3. 5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 0.5 nM to about 1 nM, about 1 nM to about 2 nM, about 2 nM to 3 nM, about 3 nM to 4 nM, about 4 nM to about 5 nM About It binds NKG2D with an affinity of K _D of 4 nM to about 10 nM, about 5 nM to about 10 nM, about 6 nM to about 10 nM, about 7 nM to about 10 nM, or about 8 nM to about 10 nM. In some embodiments, the NKG2D binding site binds NKG2D with a K _D of 10-62 nM.
CLEC12A binding site

The tumor-associated antigen binding sites of the multispecific binding proteins disclosed herein comprise a heavy chain variable domain and a light chain variable domain. Table 2 lists some exemplary sequences of heavy and light chain variable domains that in combination can bind to these tumor-associated antigens.

In one aspect, the present disclosure provides multispecific binding proteins that bind to NKG2D and CD16 receptors on natural killer cells and CLEC12A. Table 2 lists some exemplary sequences of heavy and light chain variable domains that in combination can bind to CLEC12A. CDR sequences are identified by Kabat numbering as indicated.

In certain embodiments, the second antigen binding site that binds CLEC12A (e.g., human CLEC12A) is at least 90% (e.g., at least 91%, at least 92%) the VH of an antibody disclosed in Table 2. , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) an antibody heavy chain variable domain (VH) comprising amino acid sequences that are identical, and at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, for the VL of the same antibody disclosed in Table 2) %, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the second antigen binding site is the VH and VL sequences of the antigen binding sites disclosed in Table 2, Kabat (Kabat et al., (1991) Sequences of Proteins of Immunological Interest, NIH Publication No. 91-3242, Bethesda), Chothia (see, e.g., Chothia C & Lesk A M, (1987), J Mol Biol 196:901-917), MacCallum (MacCallum R M et al., (1996) J Mol Biol 262:732-745), or any other CDR determination method known in the art. including. In certain embodiments, this second antigen-binding site comprises heavy chain CDR1, CDR2, and CDR3 and light chain CDR1, CDR2, and CDR3 of the antibodies disclosed in Table 2.

In certain embodiments, the second antigen binding site is 16B8. Derived from C8. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:135. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 136 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138, and 139, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences.

In certain embodiments, the second antigen binding site is derived from scFv-1292 or scFv-1301. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:143. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 144 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138, and 139, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is derived from scFv-1293 or scFv-1302. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:147. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 144 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138, and 139, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is derived from scFv-1294 or scFv-1303. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:244. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 144 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138, and 139, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is derived from scFv-1295 or scFv-1304. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:178. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 144 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138, and 139, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is derived from scFv-1296 or scFv-1305. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:256. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 144 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138, and 139, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is derived from scFv-1297 or scFv-1306. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:143. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 163 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138, and 139, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is derived from scFv-1298 or scFv-1307. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:143. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 167 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138, and 139, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is derived from scFv-1299 or scFv-1308. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:143. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 171 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138, and 139, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is derived from scFv-1300 or scFv-1309. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:174. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 175 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138, and 139, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is derived from scFv-1602 or scFv-2061. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:178. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:289 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138, and 139, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is humanized 16B. Derived from C8. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:182. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 151 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138, and 139, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences.

In certain embodiments, the second antigen binding site is from AB0305 or AB5030. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:270. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:271 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, A VH comprising an amino acid sequence that is at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO:336, as well as at least 90% (e.g., at least 91%, at least 92%, VL comprising amino acid sequences that are at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical. In certain embodiments, the second antigen binding site comprises VH and VL, each comprising a cysteine heterodimerization mutation. Such heterodimerization mutations can facilitate the formation of disulfide bridges between VH and VL of scFv. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 272 and 273, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 272, and 273, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is derived from AB0147 or AB7410. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:294. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:295 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 296 and 279, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 140, 141, and 142, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 296, and 279, respectively; and (b) SEQ ID NO: 140, respectively; VLs containing CDR1, CDR2, and CDR3 containing 141 and 142 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is 9F11. Derived from B7. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:193. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 194 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 192, 196 and 187, respectively. In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 198, 199 and 201, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 192, 196, and 187, respectively; and (b) SEQ ID NO: 198, respectively; VLs containing CDR1, CDR2, and CDR3 containing 199 and 201 amino acid sequences.

In certain embodiments, the second antigen binding site is from AB0191 or AB0185. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:162. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:203 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 192, 196 and 187, respectively. In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 198, 199 and 201, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 192, 196, and 187, respectively; and (b) SEQ ID NO: 198, respectively; VLs containing CDR1, CDR2, and CDR3 containing 199, and 201 amino acid sequences. In certain embodiments, the second antigen binding site is present as an scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is from AB0192 or AB0186. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:148. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:203 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 195, 196 and 187, respectively. In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 198, 199 and 201, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 195, 196, and 187, respectively; and (b) SEQ ID NO: 198, respectively; VLs containing CDR1, CDR2, and CDR3 containing 199 and 201 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is from AB0193 or AB0187. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:210. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:203 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOS: 192, 196 and 213, respectively. In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 198, 199 and 201, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 192, 196, and 213, respectively; and (b) SEQ ID NO: 198, respectively; VLs containing CDR1, CDR2, and CDR3 containing 199, and 201 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is from AB0194 or AB0188. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:162. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:218 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 192, 196 and 187, respectively. In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 198, 199 and 201, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 192, 196, and 187, respectively; and (b) SEQ ID NO: 198, respectively; VLs containing CDR1, CDR2, and CDR3 containing 199, and 201 amino acid sequences. In certain embodiments, the second antigen binding site is present as an scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is from AB0195 or AB0189. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:148. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:218 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 195, 196 and 187, respectively. In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 198, 199 and 201, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 195, 196, and 187, respectively; and (b) SEQ ID NO: 198, respectively; VLs containing CDR1, CDR2, and CDR3 containing 199, and 201 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is from AB0196 or AB0190. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:210. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:218 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 192, 196 and 213, respectively. In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 198, 199 and 201, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 192, 196, and 213, respectively; and (b) SEQ ID NO: 198, respectively; VLs containing CDR1, CDR2, and CDR3 containing 199, and 201 amino acid sequences. In certain embodiments, the second antigen binding site is present as a scFv, wherein the scFv is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences.

In certain embodiments, the second antigen binding site is humanized 9F11. Derived from B7. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:249. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:250 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOS:251, 196 and 246, respectively. In certain embodiments, the VL comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 198, 199 and 201, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 251, 196, and 246, respectively; and (b) SEQ ID NO: 198, respectively; VLs containing CDR1, CDR2, and CDR3 containing 199, and 201 amino acid sequences.

In certain embodiments, the second antigen binding site is 30A9. Derived from E9. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:247. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:248 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOS:221, 166 and 223, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:240, 226, and 179, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 221, 166, and 223, respectively; and (b) SEQ ID NO: 240, respectively; 226, and VLs containing CDR1, CDR2, and CDR3 containing 179 amino acid sequences.

In certain embodiments, the second antigen binding site is 23A5. Derived from H8. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:242. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:243 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 206, 166 and 241, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:245, 239, and 179, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising amino acid sequences of SEQ ID NOS: 206, 166, and 241, respectively; and (b) SEQ ID NO: 245, respectively; 239, and VLs containing CDR1, CDR2, and CDR3 containing 179 amino acid sequences.

In certain embodiments, the second antigen binding site is 20D6. Derived from H8. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:238. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:237 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOS:221, 236 and 223, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 152, 226, and 179, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 221, 236, and 223, respectively; and (b) SEQ ID NO: 152, respectively; 226, and VLs containing CDR1, CDR2, and CDR3 containing 179 amino acid sequences.

In certain embodiments, the second antigen binding site is 15A10. Derived from G8. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:155. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 158 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 159, 170 and 183, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 186, 188, and 189, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 159, 170, and 183, respectively; and (b) SEQ ID NO: 186, respectively; VLs containing CDR1, CDR2, and CDR3 containing 188 and 189 amino acid sequences.

In certain embodiments, the second antigen binding site is 13E1. Derived from A4. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:190. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO: 191 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 197, 202 and 207, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:211, 212, and 214, respectively. In certain embodiments, the second antigen binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 197, 202, and 207, respectively; and (b) SEQ ID NO: 211, respectively; VLs containing CDR1, CDR2, and CDR3 containing 212 and 214 amino acid sequences.

In certain embodiments, the second antigen binding site is 12F8. Derived from H7. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:217. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:219 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:220, 222, and 257, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:224, 225, and 227, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising amino acid sequences of SEQ ID NOS: 220, 222, and 257, respectively; and (b) SEQ ID NO: 224, respectively; VLs containing CDR1, CDR2, and CDR3 containing 225 and 227 amino acid sequences.

In certain embodiments, the second antigen binding site is 9E4. Derived from B7. For example, in certain embodiments, the second antigen binding site is at least 90% (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%) relative to the amino acid sequence of SEQ ID NO:228. %, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) and at least 90% (e.g., at least 91%, at least 92%) to SEQ ID NO:229 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In certain embodiments, the VH comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:230, 231, and 232, respectively. In certain embodiments, the VL comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:233, 234, and 235, respectively. In certain embodiments, the second antigen-binding site comprises (a) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:230, 231, and 232, respectively; and (b) SEQ ID NO:233, respectively; VLs containing CDR1, CDR2, and CDR3 containing 234 and 235 amino acid sequences.

In certain embodiments, the second antigen binding site that binds CLEC12A is a scFv. For example, in certain embodiments, the second antigen binding site comprises 176, 177, 180, 181, 184, 185, 204, 205, 208, 209, 215, 216, 252, 253, 254, 255, 274, 275, 280, or 281 amino acid sequences. In certain embodiments, the second antigen binding site comprises the amino acid sequence of SEQ ID NO:180.

Alternatively, novel antigen binding sites capable of binding to CLEC12A are identified by screening for binding to the amino acid sequence defined by SEQ ID NO:258, variants thereof, mature extracellular fragments thereof, or fragments containing domains of CLEC12A. obtain.
SEQ ID NO:258
MSEEVTYADLQFQNSSEMEKIPEIGKFGEKAPPAPSHVWRPAALFTLLCLLLLIGLGVL
ASMFHVTLKIEMKKMNKLQNISEELQRNISLQLMSNMNISNKIRNLSTTLQTIATKLCRE
LYSKEQEHKCKPCPRRWIWHKDSCYFLSDDVQTWQESKMACAAQNASLLKINNKNALEFIKSQSRSYDYWLGLSPEEDSTRGMRVDNIINSSAWVIRNAPDLNNMYCGYINRLYVQYYHCTYKKRMICEKMANPVQLGSTYFREA (Uniprot ID number Q5QGZ9)

It is contemplated that in scFvs, VH and VL may be connected by a linker, eg, (GlyGlyGlyGlySer) ₄ or (G ₄ S) ₄ linker (SEQ ID NO: 119). One skilled in the art will appreciate that any of the other disclosed linkers (see, e.g., Table 10) can be used in scFvs having the VH and VL sequences disclosed herein (e.g., in Table 2). will understand.

In each of the foregoing embodiments, herein the scFv, VH and/or VL sequences that bind to CLEC12A are modified by amino acid changes in the framework regions of VH and/or VL without affecting their ability to bind to CLEC12A. (eg, at least 1, 2, 3, 4, 5, or 10 amino acid substitutions, deletions, or additions). For example, as used herein, the scFv, VH and/or VL sequences that bind CLEC12A may contain cysteine heterodimerization mutations, resulting in disulfide bridge formation between the scFv VH and VL. is intended to promote

In certain embodiments, the second antigen binding site disclosed herein is measured by surface plasmon resonance (SPR) (e.g., using the method described in Example 12) or biolayer interferometry (BLI). binds human CLEC12A with a K _D (i.e., dissociation constant) of 1 nM or less, 5 nM or less, 10 nM or less, 15 nM or less, or 20 nM or less, and/or CLEC12A from a subject's bodily fluids, tissues and/or cells bind to In certain embodiments, the second antigen binding site disclosed herein is 1×10 ⁻⁵ ,1 It has a K _d (ie, off-rate, also called _Koff ) of less than or equal to × 10 ^-4 , 1 × 10 ^-3 , 5 × 10 ^-3 , 0.01, 0.02, or 0.05 1/s.

In certain embodiments, 15A10. G8 or 20D6. the second antigen binding site from A8 is 5 nM or less, 10 nM or less, as measured by surface plasmon resonance (SPR) (e.g., using the method described in Example 12) or biolayer interferometry (BLI); Binds cynomolgus monkey CLEC12A with a K _D (ie, dissociation constant) of 15 nM or less, 20 nM or less, or 30 nM or less, and/or binds CLEC12A from the body fluids, tissues and/or cells of the subject. In certain embodiments, the second antigen binding site disclosed herein is 1×10 ⁻³ , 5×10 −3 , as measured by SPR (eg, using the method described in Example 12) or BLI. It has a K _d (ie, off-rate, also called Koff) of 10 ⁻³ , 0.01, 0.02, or 0.03 1/ _s or less.

Variation in the glycosylation status of CLEC12A on the surface of different cell types has been reported (Marshall et al., (2006) Eur J Immunol. 36(8):2159-69). CLEC12A expressed on the surface of AML cells from different patients may have different glycosylation patterns as well. In addition, branched glycans can limit access to the protein component of CLEC12A and limit the diversity of available epitopes. Certain antigen binding sites of the present application can overcome glycosylation variability. In certain embodiments, a second antigen binding site disclosed herein, eg, 16B8. C8, humanized 16B8. C8, 9F11. B7, or humanized 9F11. The B7-derived antigen binding site binds CLEC12A (eg, human CLEC12A) in a glycosylation-independent manner, ie, both glycosylated and deglycosylated CLEC12A. In certain embodiments, the ratio of the _KD that binds CLEC12A in which the second antigen binding site is deglycosylated to _{the KD} that binds CLEC12A in which the second antigen binding site is glycosylated is 1: in the range of 10 to 10:1 (eg, in the range of 1:5 to 5:1, 1:3 to 3:1, or 1:2 to 2:1). In certain embodiments, the ratio is about 1:5, about 1:3, about 1:2, about 1:1.5, about 1:1, about 1.5:1, about 2:1, about 3 :1, or about 5:1. In certain embodiments, this ratio is about 1:1. In another aspect, the disclosure provides (a) a first antigen binding site that binds NKG2D; (b) a second antigen binding site that binds CLEC12A; and (c) an antibody sufficient to bind CD16. A multispecific binding protein comprising an Fc domain or portion thereof, or a third antigen binding site that binds to CD16, wherein the second antigen binding site binds to CLEC12A in a glycosylation-independent manner Join.

CLEC12A containing the K244Q substitution is a polymorphic variant prevalent in approximately 30% of the population. In some embodiments, the second antigen binding site of the multispecific binding proteins disclosed herein binds CLEC12A-K244Q. In certain embodiments, the ratio of the K _D at which the second antigen binding site binds CLEC12A-K244Q to the K _D at which the second antigen binding site binds wild-type CLEC12A is from 1:2 to 2:1. is within the range of In certain embodiments, this ratio is about 1:2, about 1:1.5, about 1:1, about 1.5:1, or about 2:1. In certain embodiments, this ratio is about 1:1.

In some embodiments, the multispecific binding proteins described herein bind CLEC12A with an isoelectric point (pI) of about 8 to about 9. In some embodiments, the multispecific binding proteins described herein bind CLEC12A with a pI of about 8.5 to about 8.9. In certain embodiments, the multispecific binding proteins described herein bind CLEC12A with a pI of about 8.7.

In some embodiments, the F3′ format of the CLEC12A TriNKET described herein (eg, hF3′-1602 TriNKET) reduces the binding to human CLEC12A on the surface of a cell, eg, a cancer cell. Format TriNKET, e.g., has an EC50 about 2 times lower than that of AB0010 (e.g., about 1 times lower, about 2 times lower, about 3 times lower, about 4 times lower, about 5 times lower, about 6 times lower) . In some embodiments, the F3′ format of CLEC12A TriNKET described herein (eg, hF3′-1602 TriNKET) is compatible with binding to human CLEC12A on the surface of cells, eg, cancer cells. about 50% lower than the EC50 of F3 format TriNKET, e.g. % lower, about 75% lower) EC50.

In certain embodiments, the second antigen binding site competes with the corresponding antigen binding site described above for binding to CLEC12A. In one aspect, the present disclosure provides (a) a first antigen binding site that binds NKG2D; (b) a second antigen binding site that binds CLEC12A; and (c) an antibody Fc sufficient to bind CD16. A multispecific binding protein is provided comprising a domain or portion thereof, or a third antigen binding site that binds CD16, wherein the second antigen binding site is, as disclosed herein, Competes with the antigen binding site for binding to CLEC12A. In certain embodiments, the second antigen-binding site is 16B8.1 disclosed above for binding to CLEC12A. It competes with the antigen binding site derived from C8. In one embodiment, the second antigen binding site is 16B8. Competes with C8. In certain embodiments, the second antigen-binding site is the humanized 16B8.1 disclosed above for binding to CLEC12A. It competes with the antigen binding site derived from C8. In one embodiment, the second antigen binding site is humanized 16B8. Competes with C8. In certain embodiments, the second antigen-binding site is 9F11.1 disclosed above for binding to CLEC12A. It competes with the antigen binding site derived from B7. In one embodiment, the second antigen binding site is 9F11. Competes with B7. In certain embodiments, the second antigen-binding site is the humanized 9F11.1 disclosed above for binding to CLEC12A. It competes with the antigen binding site derived from B7. In one embodiment, the second antigen binding site is humanized 9F11. Competes with B7. In certain embodiments, the second antigen binding site is 12F8.1 disclosed above for binding to CLEC12A. H7, 13E1. A4, 15 A10. E8, 20D6. H8, or 23A5. It competes with the antigen binding site derived from H8. In some embodiments, the second antigen binding site, for binding to CLEC12A, is 12F8. H7, 13E1. A4, 15 A10. E8, 20D6. H8, or 23A5. Competes with H8.
Fc domain

Within the Fc domain, CD16 binding is mediated by the hinge region and CH2 domain. For example, within human IgG1, interactions with CD16 are primarily through amino acid residues Asp 265-Glu 269, Asn 297-Thr 299, Ala 327-Ile 332, Leu 234-Ser 239, and carbohydrate residues in the CH2 domain. The focus is on the group N-acetyl-D-glucosamine (see Sondermann et al., Nature, 406(6793):267-273). Mutations that enhance or reduce binding affinity to CD16 may be selected based on known domains, such as using phage display libraries or yeast surface display cDNA libraries, or known domains of interaction. may be designed based on the three-dimensional structure of Thus, in certain embodiments an antibody Fc domain or portion thereof comprises a hinge and a CH2 domain.

Assembly of heterodimeric antibody heavy chains can be achieved by expressing two different antibody heavy chain sequences in the same cell, thereby allowing homodimer assembly and heterodimerization of each antibody heavy chain. assembly can result. Promotion of preferential assembly of heterodimers is disclosed in US13/494870, US16/028850, US11/533709, US12/875015, US13/289934, US14/773418, US12/811207, US13/866756, US14/647480, and This can be achieved by incorporating different mutations into the CH3 domain of each antibody heavy chain constant region, as shown in US14/830336. For example, mutations may be made in the CH3 domain based on human IgG1 to allow these two chains to preferentially heterodimerize with each other. Incorporates distinct pairs of amino acid substitutions into the polypeptide. All positions of amino acid substitution illustrated below are numbered according to the EU index as in Kabat.

In some situations, the amino acid substitution of the first polypeptide replaces the original amino acid with a larger amino acid selected from arginine (R), phenylalanine (F), tyrosine (Y), or tryptophan (W); At least one amino acid substitution in the second polypeptide replaces the original amino acid(s) with a smaller Amino acid substitutions (protrusions) that replace amino acids so that larger ones fit into the surface of smaller amino acid substitutions (cavities). For example, one polypeptide may incorporate the T366W substitution and another may incorporate three substitutions including T366S, L368A, and Y407V.

Antibody heavy chain variable domains described in this application optionally have an amino acid sequence that is at least 90% identical to an antibody constant region, such as an IgG constant region, including the hinge, CH2 and CH3 domains with or without the CH1 domain. can combine. In some embodiments, the constant region amino acid sequence is at least 90% identical to a human antibody constant region, such as a human IgG1, IgG2, IgG3, or IgG4 constant region.一実施形態では、ＣＤ１６に結合するのに十分な抗体Ｆｃドメインまたはその一部は、野生型ヒトＩｇＧ１Ｆｃ配列ＤＫＴＨＴＣＰＰＣＰＡＰＥＬＬＧＧＰＳＶＦＬＦＰＰＫＰＫＤＴＬＭＩＳＲＴＰＥＶＴＣＶＶＶＤＶＳＨＥＤＰＥＶＫＦＮＷＹＶＤＧＶＥＶＨＮＡＫＴＫＰＲＥＥＱＹＮＳＴＹＲＶＶＳＶＬＴＶＬＨＱＤＷＬＮＧＫＥＹＫＣＫＶＳＮＫＡＬＰＡＰＩＥＫＴＩＳＫＡＫＧＱＰＲＥＰＱＶＹＴＬＰＰＳＲＤＥＬＴＫＮＱＶＳＬＴＣＬＶＫＧＦＹＰＳＤＩＡＶＥＷＥＳＮＧＱＰＥＮＮＹＫＴＴＰＰＶＬＤＳＤＧＳＦＦＬＹＳＫＬＴＶＤＫＳＲＷＱＱＧＮＶＦＳＣＳＶＭＨＥＡＬＨＮＨＹＴＱＫＳＬＳＬＳＰＧ（配列番号１１８）に対して、少なくとも９０％（例えば、少なくとも９１％、少なくとも９２ %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical amino acid sequences. In some other embodiments, the constant region amino acid sequence is at least 90% identical to an antibody constant region from another mammal, such as rabbit, dog, cat, mouse, or horse.

In some embodiments, antibody constant domains linked to scFv or Fab fragments are capable of binding CD16. In some embodiments, the protein incorporates a portion of an antibody Fc domain (e.g., a portion of an antibody Fc domain sufficient to bind CD16), wherein the antibody Fc domain includes the hinge and CH2 domains ( eg, the hinge and CH2 domains of a human IgG1 antibody), and/or an amino acid sequence that is at least 90% identical to the amino acid sequence 234-332 of a human IgG antibody.

One or more mutations may be incorporated into the constant region compared to the human IgG1 constant region, e.g. K392, T394, D399, S400, D401, F405, Y407, K409, T411 and/or K439. Exemplary substitutions include, for example, Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, T350V, L351K, L351D, L351Y, S354C, E356K, E357Q, E357L, E357W, K360E, K360W, S362 、Ｓ３６４Ｅ、Ｓ３６４Ｈ、Ｓ３６４Ｄ、Ｔ３６６Ｖ、Ｔ３６６Ｉ、Ｔ３６６Ｌ、Ｔ３６６Ｍ、Ｔ３６６Ｋ、Ｔ３６６Ｗ、Ｔ３６６Ｓ、Ｌ３６８Ｅ、Ｌ３６８Ａ、Ｌ３６８Ｄ、Ｋ３７０Ｓ、Ｎ３９０Ｄ、Ｎ３９０Ｅ、Ｋ３９２Ｌ、Ｋ３９２Ｍ、Ｋ３９２Ｖ、Ｋ３９２Ｆ、Ｋ３９２Ｄ、Ｋ３９２Ｅ、Ｔ３９４Ｆ、Ｔ３９４Ｗ、Ｄ３９９Ｒ , D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, K409R, T411D, T411E, K439D, and K439E.

In certain embodiments, mutations that may be incorporated into CH1 of a human IgG1 constant region may be amino acids V125, F126, P127, T135, T139, A140, F170, P171, and/or V173. In certain embodiments, mutations that can be incorporated into Cκ of a human IgG1 constant region can be at amino acids E123, F116, S176, V163, S174, and/or T164.

Alternatively, amino acid substitutions may be selected from the following set of substitutions shown in Table 3.

Alternatively, amino acid substitutions may be selected from the following set of substitutions shown in Table 4.

Alternatively, amino acid substitutions may be selected from the following set of substitutions shown in Table 5.

Alternatively, at least one amino acid substitution in each polypeptide chain may be selected from Table 6.

Alternatively, at least one amino acid substitution may be selected from the following set of substitutions in Table 7, wherein the position(s) shown in the first polypeptide column is any known negatively charged amino acid and the position(s) shown in the second polypeptide sequence are replaced with known positively charged amino acids.

Alternatively, at least one amino acid substitution may be selected from the following set of substitutions in Table 8, wherein the position(s) indicated in the first polypeptide column is any known positively charged amino acid and the position(s) shown in the second polypeptide sequence are replaced with known negatively charged amino acids.

Alternatively, amino acid substitutions may be selected from the following set of substitutions shown in Table 9.

Alternatively, or additionally, the structural stability of the heteromultimeric protein may be increased by introducing S354C in either the first or second polypeptide chain and Y349C in the opposite polypeptide chain, This forms an artificial disulfide bridge at the interface of the two polypeptides.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at position T366, and the amino acid sequence of the other polypeptide chain of the antibody constant region is T366 , L368 and Y407.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of T366, L368 and Y407, The amino acid sequence of the other polypeptide chain of the constant region differs from that of the IgG1 constant region at position T366.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is one selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411. Different from the amino acid sequence of the IgG1 constant region at the above positions, the amino acid sequence of the other polypeptide chain of the antibody constant region is selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405 and T411. differ from the amino acid sequence of the IgG1 constant region at one or more positions indicated.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is at one or more positions selected from the group consisting of Y349, E357, S364, L368, K370, T394, D401, F405 and T411. , the amino acid sequences of the other polypeptide chains of the antibody constant region are selected from the group consisting of E357, K360, Q362, S364, L368, K370, T394, D401, F405, and T411. differ from the amino acid sequence of the IgG1 constant region at one or more positions indicated.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of L351, D399, S400 and Y407. , the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of the IgG1 constant region at one or more positions selected from the group consisting of T366, N390, K392, K409 and T411.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is combined with the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of T366, N390, K392, K409 and T411. , the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of the IgG1 constant region at one or more positions selected from the group consisting of L351, D399, S400 and Y407.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of the IgG1 constant region at one or more positions selected from the group consisting of Q347, Y349, K360, and K409. Differently, the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of the IgG1 constant region at one or more positions selected from the group consisting of Q347, E357, D399 and F405.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of Q347, E357, D399 and F405. , the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of the IgG1 constant region at one or more positions selected from the group consisting of Y349, K360, Q347 and K409.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of K370, K392, K409 and K439. , the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of the IgG1 constant region at one or more positions selected from the group consisting of D356, E357 and D399.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of D356, E357 and D399, The amino acid sequence of the other polypeptide chain of the constant region differs from that of the IgG1 constant region at one or more positions selected from the group consisting of K370, K392, K409 and K439.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region at one or more positions selected from the group consisting of L351, E356, T366 and D399. , the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of the IgG1 constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392 and K409.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region is combined with the amino acid sequence of an IgG1 constant region at one or more positions selected from the group consisting of Y349, L351, L368, K392 and K409. , the amino acid sequence of the other polypeptide chain of the antibody constant region differs from that of the IgG1 constant region at one or more positions selected from the group consisting of L351, E356, T366 and D399.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of the IgG1 constant region by a S354C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region is by a Y349C substitution. It differs from the amino acid sequence of the IgG1 constant region.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of the IgG1 constant region by a Y349C substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region differs by a S354C substitution. It differs from the amino acid sequence of the IgG1 constant region.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of the IgG1 constant region by the K360E and K409W substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region is Q347R , differs from the amino acid sequence of the IgG1 constant region by the D399V and F405T substitutions.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by the Q347R, D399V and F405T substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region is , differs from the amino acid sequence of the IgG1 constant region by the K360E and K409W substitutions.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from that of the IgG1 constant region by the T366W substitution, and the amino acid sequence of the other polypeptide chain of the antibody constant region is T366S, T368A , and differs from the amino acid sequence of the IgG1 constant region by the Y407V substitution.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by T366S, T368A, and Y407V substitutions, and the amino acid sequence of the other polypeptide chain of the antibody constant region. differs from the amino acid sequence of the IgG1 constant region by the T366W substitution.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by the T350V, L351Y, F405A, and Y407V substitutions of the other polypeptide chain of the antibody constant region. The amino acid sequence differs from that of the IgG1 constant region by the T350V, T366L, K392L and T394W substitutions.

In some embodiments, the amino acid sequence of one polypeptide chain of the antibody constant region differs from the amino acid sequence of the IgG1 constant region by the T350V, T366L, K392L, and T394W substitutions of the other polypeptide chain of the antibody constant region. The amino acid sequence differs from that of the IgG1 constant region by the T350V, L351Y, F405A and Y407V substitutions.

Exemplary Multispecific Binding Proteins Listed below are examples of TriNKETs comprising an antigen binding site that binds CLEC12A and an antigen binding site that binds NKG2D, each linked to an antibody constant region, and an antibody The constant region contains mutations that allow heterodimerization of the two Fc chains.

Exemplary CLEC12A-targeted TriNKETs are contemplated in F3' and F3 formats. As described above, in the F3' format, the antigen binding site that binds tumor-associated antigen is scFv and the antigen binding site that binds NKG2D is Fab. In the F3 format, the antigen binding site that binds tumor-associated antigen is Fab and the antigen binding site that binds NKG2D is scFv. In each TriNKET, the scFv contains a Cys substitution in the VH and VL regions to facilitate formation of a disulfide bridge between the VH and VL of the scFv.

The VH and VL of the scFv can be connected via a linker, eg, a peptide linker. In certain embodiments, a peptide linker is a flexible linker. With respect to the amino acid composition of the linker, the peptide is chosen to have properties that confer plasticity, do not interfere with the structure and function of other domains of the proteins described in this application, and resist cleavage from proteases. For example, glycine and serine residues generally provide protease resistance. In certain embodiments, VL is linked N-terminally or C-terminally to VH via a (GlyGlyGlyGlySer) ₄ ((G ₄ S) ₄ ) linker (SEQ ID NO: 119).

The length of the linker (e.g. flexible linker) may be "short", e.g. , or “long”, eg, at least 13 amino acid residues. In certain embodiments, the linker is 10-50, 10-40, 10-30, 10-25, 10-20, 15-50, 15-40, 15-30, 15-25, 15-20, 20 ~50, 20-40, 20-30, or 20-25 amino acid residues in length.

In certain embodiments, the linker is (GS) _n (SEQ ID NO: 120), (GGS) _n (SEQ ID NO: 121), (GGGS) _n (SEQ ID NO: 122), (GGSG) _n (SEQ ID NO: 123), ( GGSGG) _n (SEQ ID NO: 124) and (GGGGS) _n (SEQ ID NO: 125) sequences, wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In certain embodiments, the linker comprises or consists of an amino acid sequence selected from SEQ ID NO:119 and SEQ ID NOS:126-134, as listed in Table 10.

In F3'-TriNKET, a tumor-associated antigen-binding scFv is linked to the N-terminus of Fc via a Gly-Ser linker. Ala-Ser or Gly-Ser linkers are included in the alignment of the elbow hinge region to strike a balance between plasticity and optimal shape. In certain embodiments, an additional sequence Thr-Lys-Gly may be added at the hinge to the N-terminus or C-terminus of the Ala-Ser or Gly-Ser sequences.

As used herein to describe these exemplary TriNKETs, Fc includes antibody hinge, CH2, and CH3. In each exemplary TriNKET, the Fc domain linked to the scFv contains mutations Q347R, D399V, and F405T, and the Fc domain linked to the Fab has the matching mutation K360E to form a heterodimer. and K409W. The scFv-linked Fc domain further contains a S354C substitution in the CH3 domain, forming a disulfide bond with the Y349C substitution on the Fab-linked Fc. These permutations are in bold in the sequences described in this subsection.

For example, a TriNKET of the present disclosure is F3'-1602 TriNKET, also referred to herein as "hF3'-1602 TriNKET" and "AB0237." F3′-1602 TriNKET contains (a) the CLEC12A binding scFv sequence from scFv-1602 of Table 2, linked to the Fc domain in the N-terminal to VL direction located in VH, and (b) A49MI comprising a NKG2D-binding Fab fragment derived from a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is Connected to the Fc domain. F3'-1602 TriNKET contains three polypeptides scFv-1602-VH-VL-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL.
scFv-1602-VH-VL-Fc (SEQ ID NO:259) (“chain S”)
QLQLQESGPGLVKPSETLSLTCTVSGFSLTNYGLHWIRQPPGKCLEWIGVIWSGGKTDYNPSLKSRVTISKDTSKNQFSLKLSSVQAADTAVYYCAKYDYDDSLDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGS
DIQMTQSPSSLSASVGDRVTITCHASQNINFWLSWYQQKPGKAPKLLIYEASNLHTGVPSRFSGSGSGTRFTLTISSLQPEDIATYYCQQSHSYPLTFGCGTKLEIK
GS
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPRVYTLPPCRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLVSDGSFTLYSKLTVDKSRWQQGNVFSCSVMKSLSPGLSPGHSNHYT
A49MI-VH-CH1-Fc (SEQ ID NO:260) (“chain H”)
EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARGAPIGAAAGWFDPWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
A49MI-VL-CL (SEQ ID NO:261) (“chain L”)
DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGVSFPRTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKVYACEVTHQGLSSPVTKSFNRGEC

scFv-1602-VH-VL-Fc represents the complete sequence of clEC12A-binding scFv linked to the Fc domain via a hinge containing Gly-Ser. The Fc domain linked to the scFv contains the Q347R, D399V, and F405T substitutions for heterodimerization and the Y349C substitution at A49MI-VH-CH1-Fc to form disulfide bonds as described below. includes the S354C substitution of The scFv has the amino acid sequence of scFv-1602, which comprises the heavy chain variable domain of scFv-1602 connected via a (G ₄ S) ₄ linker to the N-terminus of the light chain variable domain of scFv-1602. include. scFv-1602-VH-VL-Fc contains Cys substitutions in the VH and VL regions of G44 and S100 to facilitate formation of disulfide bridges between scFv VH and VL.

A49MI-VH-CH1-Fc represents the heavy chain portion of the Fab fragment, comprising the heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and the CH1 domain connected to the Fc domain. The Fc domain of A49MI-VH-CH1-Fc contains the Y349C substitution of the CH3 domain and forms a disulfide bond with the S354C substitution of the Fc of scFv-1602-VH-VL-Fc. In A49MI-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization of scFv-1602-VH-VL-Fc with Fc.

A49MI-VL-CL represents the light chain portion of the Fab fragment containing the light chain constant domain of NKG2D-binding A49MI (SEQ ID NO:85) and the light chain constant domain.

The DNA sequences encoding polypeptides scFv-1602-VH-VL-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL are shown in Table 260.

In another example, a TriNKET of the present disclosure is hF3'-2061 TriNKET, also referred to herein as F3'2061 TriNKET. The F3′-2061 TriNKET contains (a) the CLEC12A binding scFv sequence from scFv-1602 of Table 2, linked to the Fc domain in the C-terminal to VL direction located in the VH, and (b) A49MI comprising a NKG2D-binding Fab fragment derived from a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is Connected to the Fc domain. F3'-2061 TriNKET contains three polypeptides scFv-2061-VL-VH-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL.
scFv-2061-VL-VH-Fc (SEQ ID NO: 262)

scFv-2061-VL-VH-Fc represents the complete sequence of tumor-associated antigen-binding scFv linked to the Fc domain via a hinge containing Gly-Ser. The Fc domain linked to the scFv contains the Q347R, D399V, and F405T substitutions for heterodimerization and the S354C substitution to form a disulfide bond with the Y349C substitution at A49MI-VH-CH1-Fc below. Included as explained. The scFv has the amino acid sequence of scFv-2061, comprising the heavy chain variable domain of scFv-2061 connected via a (G ₄ S) ₄ linker to the C-terminus of the light chain variable domain of scFv-2061. included. It is contemplated that scFv-2061-VL-VH-Fc may contain Cys substitutions in the VH and VL regions that facilitate formation of disulfide bridges between the VH and VL of the ScFv.

A49MI-VH-CH1-Fc represents the heavy chain portion of the Fab fragment, comprising the heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and the CH1 domain connected to the Fc domain. The Fc domain of A49MI-VH-CH1-Fc contains the Y349C substitution of the CH3 domain and forms a disulfide bond with the S354C substitution of the Fc of scFv-2061-VL-VH-Fc. In A49MI-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization of scFv-2061-VL-VH-Fc with Fc.

A49MI-VL-CL represents the light chain portion of the Fab fragment containing the light chain constant domain of NKG2D-binding A49MI (SEQ ID NO:85) and the light chain constant domain.

In another example, the TriNKET of the present disclosure is AB0089TriNKET. AB0089 TriNKET contains (a) the CLEC12A-binding scFv sequence from AB0305 of Table 2 linked to the Fc domain in the N-terminal to VL direction located in the VH and (b) the NKG2D-binding Fab from A49MI fragments, comprising a heavy chain portion comprising the heavy chain variable domain and the CH1 domain, and a light chain portion comprising the light chain variable domain and the light chain constant domain, wherein the CH1 domain is connected to the Fc domain ing. AB0089 TriNKET contains three polypeptides AB0305-VH-VL-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL.
AB0305-VH-VL-Fc (SEQ ID NO:276)

AB0305-VH-VL-Fc represents the complete sequence of tumor-associated antigen-binding scFv linked to the Fc domain via a hinge containing Gly-Ser. The Fc domain linked to the scFv contains the Q347R, D399V, and F405T substitutions for heterodimerization and the S354C substitution to form a disulfide bond with the Y349C substitution at A49MI-VH-CH1-Fc below. Included as explained. The scFv contains the heavy chain variable domain of AB0305 connected via a (G ₄ S) ₄ linker to the N-terminus of the light chain variable domain of AB0305. AB0305-VH-VL-Fc contains Cys substitutions in the VH and VL regions of G44 and S100 to facilitate the formation of disulfide bridges between the scFv VH and VL.

A49MI-VH-CH1-Fc represents the heavy chain portion of the Fab fragment, comprising the heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and the CH1 domain connected to the Fc domain. The Fc domain of A49MI-VH-CH1-Fc contains a Y349C substitution in the CH3 domain that forms a disulfide bond with the S354C substitution on the Fc of AB0305-VH-VL-Fc. In A49MI-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization with Fc of AB0305-VH-VL-Fc.

A49MI-VL-CL represents the light chain portion of the Fab fragment containing the light chain constant domain of NKG2D-binding A49MI (SEQ ID NO:85) and the light chain constant domain.

In another example, a TriNKET of the present disclosure is AB0147 TriNKET. AB0147 TriNKET contains (a) the CLEC12A-binding scFv sequence from AB0304 of Table 2, linked to the Fc domain in the N-terminal to VL direction located in the VH, and (b) the NKG2D-binding Fab from A49MI fragments, comprising a heavy chain portion comprising the heavy chain variable domain and the CH1 domain, and a light chain portion comprising the light chain variable domain and the light chain constant domain, wherein the CH1 domain is connected to the Fc domain ing. AB0147 TriNKET contains three polypeptides AB0304-VH-VL-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL.
AB0304-VH-VL-Fc (SEQ ID NO:277)

AB0304-VH-VL-Fc represents the complete sequence of tumor-associated antigen-binding scFv linked to the Fc domain via a hinge containing Gly-Ser. The Fc domain linked to the scFv has Q347R, D399V, and F405T substitutions for heterodimerization and a S354C substitution to form a disulfide bond with the Y349C substitution at A49MI-VH-CH1-Fc below. Included as explained. The scFv contains the heavy chain variable domain of AB0304 connected to the N-terminus of the light chain variable domain of AB0304 via a (G ₄ S) ₄ linker. AB0304-VH-VL-Fc contains Cys substitutions at G44 and S100 in the VH and VL regions that promote formation of disulfide bridges between the VH and VL of ScFv.

A49MI-VH-CH1-Fc represents the heavy chain portion of the Fab fragment, comprising the heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and the CH1 domain connected to the Fc domain. The Fc domain of A49MI-VH-CH1-Fc contains a Y349C substitution in the CH3 domain that forms a disulfide bond with the S354C substitution on the Fc of AB0037-VH-VL-Fc. In A49MI-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization with Fc of AB0305-VH-VL-Fc.

A49MI-VL-CL represents the light chain portion of the Fab fragment containing the light chain constant domain of NKG2D-binding A49MI (SEQ ID NO:85) and the light chain constant domain.

In another example, the TriNKET of the present disclosure is AB0192 TriNKET. The AB0192 TriNKET contains (a) the CLEC12A-binding scFv sequence from AB0192 of Table 2, linked to the Fc domain in the N-terminal to VL direction located in the VH, and (b) the NKG2D-binding Fab from A49MI. fragments, comprising a heavy chain portion comprising the heavy chain variable domain and the CH1 domain, and a light chain portion comprising the light chain variable domain and the light chain constant domain, wherein the CH1 domain is connected to the Fc domain ing. AB0192 TriNKET contains three polypeptides AB0192-VH-VL-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL.
AB0192-VH-VL-Fc (SEQ ID NO:278)

AB0192-VH-VL-Fc represents the complete sequence of tumor-associated antigen-binding scFv linked to the Fc domain via a hinge containing Gly-Ser. The Fc domain linked to the scFv has Q347R, D399V, and F405T substitutions for heterodimerization and a S354C substitution to form a disulfide bond with the Y349C substitution at A49MI-VH-CH1-Fc below. Included as explained. The scFv contains the heavy chain variable domain of AB0192 connected via a (G ₄ S) ₄ linker to the N-terminus of the light chain variable domain of AB0192. AB0192-VH-VL-Fc contains Cys substitutions at G44 and S100 in the VH and VL regions that promote the formation of disulfide bridges between the VH and VL of ScFv.

A49MI-VH-CH1-Fc represents the heavy chain portion of the Fab fragment, comprising the heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and the CH1 domain connected to the Fc domain. The Fc domain of A49MI-VH-CH1-Fc contains a Y349C substitution in the CH3 domain that forms a disulfide bond with the S354C substitution on the Fc of AB0192-VH-VL-Fc. In A49MI-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization with Fc of AB0192-VH-VL-Fc.

A49MI-VL-CL represents the light chain portion of the Fab fragment containing the light chain constant domain of NKG2D-binding A49MI (SEQ ID NO:85) and the light chain constant domain.

In another example, a TriNKET of the present disclosure is hF3'-1295 TriNKET, also referred to herein as F3'-1295 TriNKET. F3'-1295 TriNKET contains (a) the CLEC12A binding scFv sequence from scFv-1295 of Table 2, linked to the Fc domain in the N-terminal to VL direction located in VH, and (b) A49MI comprising a NKG2D-binding Fab fragment derived from a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is Connected to the Fc domain. F3'-1295 TriNKET contains three polypeptides scFv-1295-VH-VL-Fc, A49MI-VH-CH1-Fc, and A49MI-VL-CL.
scFv-1295-VH-VL-Fc (SEQ ID NO:281)

scFv-1295-VH-VL-Fc represents the complete sequence of tumor-associated antigen-binding scFv linked to the Fc domain via a hinge containing Gly-Ser. The Fc domain linked to the scFv contains the Q347R, D399V, and F405T substitutions for heterodimerization and the S354C substitution to form a disulfide bond with the Y349C substitution at A49MI-VH-CH1-Fc below. Included as explained. The scFv comprises the heavy chain variable domain of scFv-1295 connected via a (G ₄ S) ₄ linker to the N-terminus of the light chain variable domain of scFv-1295. scFv-1295-VH-VL-Fc contains Cys substitutions at G44 and S100 of the VH and VL regions to facilitate formation of disulfide bridges between VH and VL of the scFv.

A49MI-VH-CH1-Fc represents the heavy chain portion of the Fab fragment, comprising the heavy chain variable domain of NKG2D-binding A49MI (SEQ ID NO:95) and the CH1 domain connected to the Fc domain. The Fc domain of A49MI-VH-CH1-Fc contains a Y349C substitution in the CH3 domain that forms a disulfide bond with the S354C substitution on the Fc of AB0192-VH-VL-Fc. In A49MI-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization of scFv-1295-VH-VL-Fc with Fc.

A49MI-VL-CL represents the light chain portion of the Fab fragment containing the light chain constant domain of NKG2D-binding A49MI (SEQ ID NO:85) and the light chain constant domain.

1602-VH-CH1-Fc（配列番号287)
QLQLQESGPGLVKPSETLSLTCTVSGFSLTNYGLHWIRQPPGKGLEWIGVIWSGGKTDYNPSLKSRVTISKDTSKNQFSLKLSSVQAADTAVYYCAKYDYDDSLDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
1602－VL－CL（配列番号288)
DIQMTQSPSSLSASVGDRVTITCHASQNINFWLSWYQQKPGKAPKLLIYEASNLHTGVPSRFSGSGSGTRFTLTISSLQPEDIATYYCQQSHSYPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC In another example, the TriNKET of the present disclosure is F3 1602 TriNKET, also referred to herein as AB0010. F3 1602 TriNKET contains (a) the NKG2D-binding scFv sequence from scFv-A49MI of Table 1, linked to the Fc domain in the N-terminal to VL direction located in the VH, and (b) scFv-1602 comprising a CLEC12A-binding Fab fragment derived from a heavy chain portion comprising a heavy chain variable domain and a CH1 domain, and a light chain portion comprising a light chain variable domain and a light chain constant domain, wherein the CH1 domain is Connected to the Fc domain. F3 1602 TriNKET contains three polypeptides scFv-A49MI-VH-VL-Fc, 1602-VH-CH1-Fc, and 1602-VL-CL.
scFv-A49MI-VH-VL-Fc (SEQ ID NO:286)

1602-VH-CH1-Fc (SEQ ID NO:287)
QLQLQESGPGLVKPSETLSLTCTVSGFSLTNYGLHWIRQPPGKGLEWIGVIWSGGKTDYNPSLKSRVTISKDTSKNQFSLKLSSVQAADTAVYYCAKYDYDDSLDYWGQGTLVTVSS
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTENQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSWLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
1602-VL-CL (SEQ ID NO: 288)
DIQMTQSPSSLSASVGDRVTITCHASQNINFWLSWYQQKPGKAPKLLIYEASNLHTGVPSRFSGSGSGTRFTLTISSLQPEDIATYYCQQSHSYPLTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

scFv-A49MI-VH-VL-Fc represents the complete sequence of NKG2D-binding scFv A49MI linked to the Fc domain via a hinge containing Gly-Ser. The Fc domain linked to the scFv contains the Q347R, D399V, and F405T substitutions for heterodimerization and the Y349C substitution at A49MI-VH-CH1-Fc to form disulfide bonds as described below. includes the S354C substitution of The scFv contains the heavy chain variable domain of scFv-1295 connected via a (G ₄ S) ₄ linker to the N-terminus of the light chain variable domain of scFv-A49MI. scFv-A49MI-VH-VL-Fc contains Cys substitutions at G44 and S100 of the VH and VL regions to facilitate formation of disulfide bridges between VH and VL of the scFv.

1602-VH-CH1-Fc represents the heavy chain portion of the Fab fragment and contains the CH1 domain connected to the heavy chain variable domain and Fc domain of CLEC12A binding 1602. The Fc domain of 1602-VH-CH1-Fc contains the Y349C substitution of the CH3 domain and forms a disulfide bond with the S354C substitution of the Fc of scFv-A49MI-VH-VL-Fc. In 1602-VH-CH1-Fc, the Fc domain also contains K360E and K409W substitutions for heterodimerization of scFv-A49MI-VH-VL-Fc with Fc.

1602-VL-CL represents the light chain portion of the Fab fragment containing the light chain variable and light chain constant domains of CLEC12A binding 1602.

In certain embodiments, the TriNKETs of the disclosure are wherein the Fc domain linked to the NKG2D-binding Fab fragment comprises Q347R, D399V, and F405T substitutions and the Fc domain linked to the tumor-associated antigen-binding scFv comprises heterodimeric Identical to one of the exemplary TriNKETs described above containing the EW-RVT Fc mutation, except that it contains the matching substitutions K360E and K409W to form mers. In certain embodiments, the TriNKETs of this disclosure are those in which the Fc domain linked to the NKG2D-binding Fab fragment comprises a "hole" substitution of T366S, L368A, and Y407V, and the Fc domain linked to the tumor-associated antigen-binding scFv is , identical to one of the above exemplary TriNKETs containing the KiH Fc mutation, except that it contains a "knob" substitution of T366W to form a heterodimer.

In certain embodiments, the TriNKETs of the present disclosure are such that the Fc domain linked to the NKG2D-binding Fab fragment comprises a S354C substitution in the CH3 domain, and the Fc domain linked to the tumor-associated antigen-binding scFv forms a disulfide bond. Identical to one of the exemplary TriNKETs above, except that it contains a matching Y349C substitution in the CH3 domain to

Those skilled in the art recognize that during production and/or storage of a protein, N-terminal glutamic acid (E) or glutamine (Q) (e.g., spontaneously or catalyzed by enzymes present during production and/or storage) It will be appreciated that it can be cyclized to form a lactam. Thus, in some embodiments where the N-terminal residue of the amino acid sequence of the polypeptide is E or Q, the corresponding amino acid sequences in which E or Q are replaced with pyroglutamic acid are also contemplated herein.

One skilled in the art will also appreciate that the C-terminal lysine (K) of a protein may be removed during production and/or storage of the protein (e.g., spontaneously or during production and/or storage). catalyzed by existing enzymes). Such K removal is often observed in proteins containing an Fc domain at the C-terminus. Thus, in some embodiments where the C-terminal residue of a polypeptide amino acid sequence (eg, an Fc domain sequence) is a K, the corresponding amino acid sequence with the K removed is also contemplated herein.

The multispecific proteins described above can be made using recombinant DNA technology well known to those skilled in the art. For example, a first nucleic acid sequence encoding a first immunoglobulin heavy chain may be cloned into a first expression vector; a second nucleic acid sequence encoding a second immunoglobulin heavy chain may be cloned into a second a third nucleic acid sequence encoding an immunoglobulin light chain may be cloned into a third expression vector; and the first, second, and third expression vectors may be stably transfected together into a host cell to produce multimeric proteins.

To achieve the highest yield of multispecific protein, different ratios of the first, second and third expression vectors can be tested to determine the optimal ratio for transfection into host cells. After transfection, single clones can be isolated for cell bank generation using methods known in the art such as limiting dilution, ELISA, FACS, microscopy, or Clonepix.

The application also provides nucleic acids that encode polypeptides that are part of the multispecific proteins provided herein. In some embodiments, a multispecific protein comprises three polypeptides. In some embodiments, the multispecific protein comprises (a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:276; (b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:260; and (c ) a third polypeptide comprising the amino acid sequence of SEQ ID NO:261; In certain embodiments, the application provides nucleic acids encoding each of the three polypeptides that are part of the multispecific protein. In certain embodiments, provided herein is a nucleic acid encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO:276. In certain embodiments, provided herein is a nucleic acid encoding a second polypeptide comprising the amino acid sequence of SEQ ID NO:260. In certain embodiments, provided herein is a nucleic acid encoding a third polypeptide comprising the amino acid sequence of SEQ ID NO:261.

In another aspect, the application provides cells comprising one or more nucleic acids encoding polypeptides that are part of the multispecific proteins provided herein. In some embodiments, the cell contains one or more nucleic acids that encode one or more of the three polypeptides that are each part of the multispecific protein. In certain embodiments, provided herein are cells comprising a nucleic acid encoding a first polypeptide comprising the amino acid sequence of SEQ ID NO:276. In certain embodiments, provided herein is a cell comprising a nucleic acid encoding a second polypeptide comprising the amino acid sequence of SEQ ID NO:260. In certain embodiments, provided herein is a cell comprising a nucleic acid encoding a third polypeptide comprising the amino acid sequence of SEQ ID NO:261.

Also provided herein are methods of producing or making the multispecific proteins provided herein. In some embodiments, the method comprises: (a) culturing a host cell under conditions suitable for expression of the protein, wherein the host cell is a multispecific protein (b) isolating and purifying the protein; In some embodiments, the method comprises (a) culturing a host cell under conditions suitable for expression of the protein, wherein the host cell comprises a first poly(1) comprising the amino acid sequence of SEQ ID NO:276; A first nucleic acid encoding a peptide, a second nucleic acid encoding a second polypeptide comprising the amino acid sequence of SEQ ID NO:260, and a third nucleic acid encoding a third polypeptide comprising the amino acid sequence of SEQ ID NO:261 comprising the nucleic acid; and (b) isolating and purifying the protein.

Clones can be cultured under conditions suitable for bioreactor scale-up and maintenance of multispecific protein expression. Multispecific proteins can be analyzed using techniques of the art including centrifugation, depth filtration, cell lysis, homogenization, freeze-thaw, affinity purification, gel filtration, ion exchange chromatography, hydrophobic interaction exchange chromatography, and mixed mode chromatography. It can be isolated and purified using methods known in the art.

II. Features of Multispecific Proteins The multispecific proteins described herein include a NKG2D binding site, a tumor-associated antigen binding site that binds CLEC12A, and an antibody Fc domain or portion thereof sufficient to bind CD16; or an antigen binding site that binds to CD16. In some embodiments, the multispecific protein comprises additional antigen binding sites that bind the same tumor-associated antigen (CLEC12A), as illustrated in the F4-TriNKET format (eg, Figures 2C and 2D).

In some embodiments, the multispecific protein exhibits thermal stability similar to a corresponding monoclonal antibody, ie, a monoclonal antibody containing the same tumor-associated antigen binding site incorporated into the multispecific protein.

In some embodiments, the multispecific protein simultaneously binds to cells expressing NKG2D and/or CD16, eg, NK cells, and cells expressing CLEC12A, eg, certain tumor cells. Binding of multispecific proteins to NK cells can enhance the activity of NK cells toward destruction of tumor cells expressing tumor-associated antigens. NK cells have been reported to exhibit more potent cytotoxicity against stressed target cells (see Chan et al., (2014) Cell Death Differ 21(1):5-14). ). Without wishing to be bound by theory, when NK cells are engaged to a population of cells by TriNKET, they select stressed target cells (e.g., malignant cells and cells within the tumor microenvironment). presumed to be able to kill This mechanism may contribute to the increased specificity and reduced toxicity of TriNKET, allowing selective elimination of stressed cells, even though expression of tumor-associated antigens is not restricted to desired target cells. enable

In some embodiments, this multispecific protein induces antibody-dependent cellular phagocytosis (ADCP). In some embodiments, the multispecific protein induces antibody-dependent cellular cytotoxicity (ADCC)-like activity. In some embodiments, the multispecific protein induces ADCC activity. In some embodiments, the multispecific protein does not induce complement dependent cytotoxicity (CDC).

In some embodiments, the multispecific protein has similar affinity to a corresponding anti-tumor-associated antigen monoclonal antibody (i.e., a monoclonal antibody comprising the same tumor-associated antigen binding site incorporated into the multispecific protein). Binds tumor-associated antigens. In some embodiments, the multispecific proteins are more effective at killing tumor cells expressing tumor-associated antigens than the corresponding monoclonal antibodies.

In certain embodiments, a multispecific protein described herein comprising a binding site for a tumor-associated antigen activates primary human NK cells when co-cultured with cells expressing that tumor-associated antigen. NK cell activation is characterized by CD107a degranulation and increased IFN-γ cytokine production. Furthermore, compared to the corresponding anti-tumor-associated antigen monoclonal antibodies, the multispecific proteins may exhibit superior activation of human NK cells in the presence of cells expressing tumor-associated antigens.

In some embodiments, a multispecific protein described herein comprising a binding site for a tumor-associated antigen is associated with resting and IL-2-activated human NK cells when co-cultured with cells expressing the tumor-associated antigen. enhances the activity of

In some embodiments, multispecific proteins provide advantages in targeting tumor cells that express moderate and low levels of tumor-associated antigens compared to corresponding monoclonal antibodies that bind tumor-associated antigens. do.

In some embodiments, the bivalent F4 format of TriNKET (i.e., TriNKET contains additional antigen binding sites that bind tumor-associated antigens) improves the avidity of TriNKET binding to tumor-associated antigens, which is In effect, it stabilizes the expression and maintenance of high levels of tumor-associated antigens on the surface of tumor cells. In some embodiments, the F4-TriNKET mediates more potent tumor cell killing than the corresponding F3-TriNKET or F3'-TriNKET.

III. Pharmaceutical Formulations The present disclosure also provides pharmaceutical formulations comprising a therapeutically effective amount of a multispecific binding protein (eg, F3'-1602) disclosed herein. The pharmaceutical formulation contains one or more excipients and is maintained at a particular pH. As used herein, the term "excipient" refers to a desired physical or chemical property such as pH, osmotic pressure, viscosity, clarity, color, isotonicity, odor, sterility, stability, It means any non-therapeutic agent added to the formulation to provide a rate of dissolution or release, adsorption, or penetration.

Excipients and pH
One or more excipients in the pharmaceutical formulations of the polyspecific binding proteins described in this application include buffering agents. As used herein, the term "buffering agent" refers to one or more buffers that, when added to an aqueous solution, can protect the solution from pH fluctuations when acid or alkali is added, or when diluted with a solvent. point to the ingredients. In addition to phosphate buffers, glycinate, carbonate, citrate, histidine buffers, etc. may be used, in which case sodium, potassium, or ammonium ions may serve as counterions.

In certain embodiments, the buffer or buffer system comprises at least one buffer having a buffer range that fully or partially overlaps the pH range of 7.0-9.0. In certain embodiments, the buffer has a pKa of about 7.0±0.5. In certain embodiments, the buffer has a pKa of about 7.5±0.5.

In certain embodiments, the buffer comprises citrate buffer. In certain embodiments, the citrate is 5-100 mM, 10-100 mM, 15-100 mM, 20-100 mM, 5-50 mM, 10-50 mM, 15-100 mM, 20-100 mM, 5-25 mM, 10-25 mM , 15-25 mM, 20-25 mM, 5-20 mM, 10-20 mM, or 15-20 mM. In certain embodiments, citrate is present at a concentration of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 50 mM. In certain embodiments, citrate is present at a concentration of 20 mM.

In certain embodiments, the buffer comprises or further comprises a phosphate buffer. In certain embodiments, the phosphate is 5-100 mM, 10-100 mM, 15-100 mM, 20-100 mM, 5-50 mM, 10-50 mM, 15-100 mM, 20-100 mM, 5-25 mM, 10-25 mM , 15-25 mM, 20-25 mM, 5-20 mM, 10-20 mM, or 15-20 mM. In certain embodiments, phosphate is present at a concentration of 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, or 50 mM. In certain embodiments, phosphate is present at a concentration of 10 mM. It is understood that phosphates are not readily compatible with lyophilized presentations. Thus, in certain embodiments, the concentration of phosphate, if any, in the pharmaceutical formulation is 5 mM, 4 mM, 3 mM, 2 mM, 1 mM, 0.5 mM, 0.1 mM, 50 μM, 10 μM, 5 μM, or 1 μM or less. In certain embodiments, the concentration of phosphate in the pharmaceutical formulation is below the limit of detection. In certain embodiments, no phosphate is added when preparing the pharmaceutical formulation.

Pharmaceutical formulations of the polyspecific binding proteins described in this application can have a pH greater than 7.0. For example, in certain embodiments, the pharmaceutical formulation has a 7.0-7.6 (ie 7.3±0.3), 7.0-7.4 (ie 7.2±0.2), 7.0-7.2 (ie 7. 1±0.1), 7.1-7.3 (ie 7.2±0.1), 7.2-7.4 (ie 7.3±0.1), 7.3-7 .5 (ie 7.4±0.1), 7.4-7.6 (ie 7.5±0.1), 7.5-7.7 (ie 7.6±0.1 ), 7.6-7.8 (i.e. 7.7±0.1), 7.7-7.9 (i.e. 7.8±0.1), or 7.8-8.0 (i.e. , 7.9±0.1). In certain embodiments, the pharmaceutical formulation comprises 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or has a pH of 8.0. In certain embodiments, the pharmaceutical formulation has a pH of 7.0. The scientific rounding rule is that a pH greater than or equal to 6.95 and less than or equal to 7.05 is rounded as 7.0.

One or more excipients in pharmaceutical formulations of the polyspecific binding proteins described in this application further include sugars and sugar alcohols. Sugars and sugar alcohols are useful in pharmaceutical formulations as heat stabilizers. In certain embodiments, pharmaceutical formulations comprise sugars, such as monosaccharides (glucose, xylose, or erythritol), disaccharides (e.g., sucrose, trehalose, maltose, or galactose), or oligosaccharides (e.g., stachyose). . In certain embodiments, the pharmaceutical formulation comprises sucrose. In certain embodiments, the pharmaceutical composition comprises a sugar alcohol, such as a sugar alcohol derived from a monosaccharide (eg, mannitol, sorbitol, or xylitol), a sugar alcohol derived from a disaccharide (eg, lactitol or maltitol), or sugar alcohols derived from oligosaccharides. In certain embodiments, the pharmaceutical formulation comprises sorbitol.

The amount of sugar or sugar alcohol included in the formulation may vary depending on the particular situation and intended purpose in which the formulation is used. In certain embodiments, the pharmaceutical formulation comprises 50-300 mM, 50-250 mM, 100-300 mM, 100-250 mM, 150-300 mM, 150-250 mM, 200-300 mM, 200-250 mM, or 250-300 mM sugar or sugar Contains alcohol. In certain embodiments, the pharmaceutical formulation comprises 50 mM, 75 mM, 100 mM, 125 mM, 150 mM, 200 mM, 250 mM, or 300 mM sugar or sugar alcohol. In certain embodiments, the pharmaceutical formulation contains 250 mM sugar or sugar alcohol (eg, sucrose or sorbitol).

One or more excipients in the pharmaceutical formulations disclosed herein further comprise a surfactant. As used herein, the term "surfactant" refers to surface-active molecules that contain both hydrophobic moieties (eg, alkyl chains) and hydrophilic moieties (eg, carboxyl and carboxylate groups). Surfactants are useful in pharmaceutical formulations to reduce aggregation of therapeutic proteins. Surfactants suitable for use in pharmaceutical formulations are generally non-ionic surfactants, including but not limited to polysorbates (e.g. polysorbate 20 or polysorbate 80); poloxamers (e.g. poloxamer 188); sorbitan sodium lauryl sulfate; sodium octylglucoside; laurylsulfobetaine, myristylsulfobetaine, linoleylsulfobetaine, or stearylsulfobetaine; laurylsarcosine, myristylsarcosine, linoleylsarcosine, or stearylsarcosine; linoleylbetaine, myristyl betaine, or cetyl betaine; lauroamidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl betaine, myristamidopropyl betaine, palmidopropyl betaine, or isostearamidopropyl betaine (e.g. lauroamidopropyl); myristamidopropyl dimethylamine , palmidopropyl dimethylamine, or isostearamidopropyl dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; and the MONAQUAT™ series (Mona Industries, Inc., Paterson, N.J.), polyethyl Glycols, polypropyl glycols, and copolymers of ethylene glycol and propylene glycol (eg, Pluronics, PF68, etc.). In certain embodiments, the surfactant is polysorbate. In certain embodiments, the surfactant is polysorbate 80.

The amount of nonionic surfactant included within the pharmaceutical formulations of the multispecific binding proteins described in this application will depend on the particular properties desired in the formulation, as well as the particular circumstances and purposes for which the formulation is intended to be used. can change. In certain embodiments, the formulation contains 0.005%-0.5%, 0.005%-0.2%, 0.005%-0.1%, 0.005%-0.05%, 0 0.005%-0.02%, 0.005%-0.01%, 0.01%-0.5%, 0.01%-0.2%, 0.01%-0.1%, 0 0.01% to 0.05%, or 0.01% to 0.02% of a nonionic surfactant (eg, polysorbate 80). In certain embodiments, the pharmaceutical formulation contains 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.15%, 0.2%, 0.25%, 0.3%, 0.35%, 0.4%, 0.45%, or 0.5% non-ionic surfactant (eg polysorbate 80).

In certain embodiments, pharmaceutical formulations are isotonic. An "isotonic" formulation is one that has essentially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of about 250-350 mOsmol/kg _H2O . Isotonicity can be measured using a vapor pressure or ice osmometer. In certain embodiments, the pharmaceutical formulation has an osmolality of 250-350 mOsmol/kg H ₂ O. In certain embodiments, the pharmaceutical formulation has an osmolality of 300-350 mOsmol/kg H ₂ O.

Agents such as sugars, sugar alcohols, and NaCl may be included in pharmaceutical formulations for desirable tonicity. It is understood that NaCl is not readily compatible with lyophilized presentation. Thus, in certain embodiments, the concentration of NaCl, if any, in the pharmaceutical formulation is , 10 μM, 5 μM, or 1 μM or less. In certain embodiments, the concentration of NaCl in the pharmaceutical formulation is below the limit of detection. In certain embodiments, no NaCl salt is added when preparing the pharmaceutical formulation.

Pharmaceutical formulations of the multispecific binding proteins described in this application may further comprise one or more other substances such as bulking agents or preservatives. A "bulking agent" is a compound that adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates the production of an essentially uniform lyophilized cake that maintains an open pore structure). is. Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. Lyophilized formulations of polyspecific binding proteins described in this application may include such bulking agents. Preservatives reduce bacterial action and can, for example, facilitate the manufacture of multi-use (multi-dose) formulations.

Exemplary Formulations In certain embodiments, pharmaceutical formulations of the multispecific binding proteins described in this application contain multispecific binding agents in a buffer of pH 7.0 or higher (eg, pH 7.0-8.0). Contains protein.

In certain embodiments, the pharmaceutical formulation comprises 10-200 mg/mL multispecific binding protein, 10-25 mM citrate, 200-300 mM sugar or sugar alcohol (eg, sucrose), and 0.005% ˜0.05% polysorbate (eg, polysorbate 80), pH 7.0-8.0. In certain embodiments, the pharmaceutical formulation contains 10-200 mg/mL multispecific binding protein, 20 mM citrate, 250 mM sugar or sugar alcohol (eg, sucrose), and 0.01% polysorbate (eg, , polysorbate 80) at pH 7.0-8.0. In certain embodiments, the pharmaceutical formulation contains 10-200 mg/mL multispecific binding protein, 20 mM citrate, 250 mM sugar or sugar alcohol (eg, sucrose), and 0.01% polysorbate (eg, , polysorbate 80) at pH 7.0-7.5 (eg, pH 7.0).

In certain embodiments, the pharmaceutical formulation comprises 10-50 mg/mL multispecific binding protein, 10-25 mM citrate, 200-300 mM sugar or sugar alcohol (eg, sucrose), and 0.005% ˜0.05% polysorbate (eg, polysorbate 80) at pH 7.0-8.0. In certain embodiments, the pharmaceutical formulation contains 10-50 mg/mL multispecific binding protein, 20 mM citrate, 250 mM sugar or sugar alcohol (eg, sucrose), and 0.01% polysorbate (eg, , polysorbate 80) at pH 7.0-8.0. In certain embodiments, the pharmaceutical formulation contains 10-50 mg/mL multispecific binding protein, 20 mM citrate, 250 mM sugar or sugar alcohol (eg, sucrose), and 0.01% polysorbate (eg, , polysorbate 80) at pH 7.0-7.5 (eg, pH 7.0).

In certain embodiments, the pharmaceutical formulation contains 10-200 mg/mL multispecific binding protein, 5-20 mM citrate, 5-20 mM phosphate, and 100-150 mM NaCl, pH 7.0. Including at ~8.0. In certain embodiments, the pharmaceutical formulation comprises 10-200 mg/mL multispecific binding protein, 10 mM citrate, 10 mM phosphate, and 125 mM NaCl at pH 7.0-8.0. . In certain embodiments, the pharmaceutical formulation comprises 10-200 mg/mL multispecific binding protein, 10 mM citrate, 10 mM phosphate, and 125 mM NaCl at pH 7.5-8.0 (eg , pH 7.8).

In certain embodiments, the pharmaceutical formulation contains 10-50 mg/mL multispecific binding protein, 5-20 mM citrate, 5-20 mM phosphate, and 100-150 mM NaCl, pH 7.0. Including at ~8.0. In certain embodiments, the pharmaceutical formulation comprises 10-50 mg/mL multispecific binding protein, 10 mM citrate, 10 mM phosphate, and 125 mM NaCl at pH 7.0-8.0. . In certain embodiments, the pharmaceutical formulation comprises 10-50 mg/mL multispecific binding protein, 10 mM citrate, 10 mM phosphate, and 125 mM NaCl at pH 7.5-8.0 (eg , pH 7.8).

Stability of Multispecific Binding Proteins Pharmaceutical formulations of multispecific binding proteins described in this application exhibit a high level of stability. A pharmaceutical formulation is stable if the polyspecific binding proteins within it retain an acceptable degree of physical properties, chemical structure and/or biological function after storage under defined conditions. are doing.

Stresses during manufacture, storage, and use of this pharmaceutical formulation include, but are not limited to, heat stress (e.g., incubation at 40°C for 4 weeks), freeze/thaw stress (e.g., freeze/thaw 6 times). cycle), agitation stress (e.g., 60 rpm, 18 h agitation at 4°C), low pH stress (e.g., pH 5.0, 2 weeks incubation at 40°C), high pH stress (e.g., pH 8.0, 40°C). 2 weeks incubation at room temperature), oxidative stress (e.g. forced oxidation with 0.02% hydrogen peroxide at room temperature for 24 hours), or low pH holding (e.g. incubation at pH 3.3 for 1.5 hours at room temperature). is mentioned. In certain embodiments, multispecific binding proteins are stable following exposure to one or more of these stresses.

Stability can be measured by determining the percentage of multispecific binding protein in a formulation that remains in its native conformation after storage at a defined temperature for a defined period of time. The percentage of protein in the native conformation can be determined, for example, by size exclusion chromatography (e.g., size exclusion high performance liquid chromatography), wherein the protein in the native conformation is not aggregated (in the high molecular weight fraction). eluted) or not degraded (neither eluted into low molecular weight fractions). In certain embodiments, less than 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% of the multispecific binding proteins disclosed herein After one or more stresses, it forms high molecular weight complexes (ie, having a higher molecular weight than the native protein) as determined by size exclusion chromatography. In certain embodiments, less than 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% of the multispecific binding proteins disclosed herein After one or more stresses, it is degraded (ie, has a lower molecular weight than the native protein), as determined by size exclusion chromatography.

Stability is also measured by determining the percentage of multispecific binding protein present in a more acidic fraction (the "acidic form") compared to the major fraction of the protein (the "major charge form"). can be measured. Without wishing to be bound by theory, deamidation of a protein may render it more negatively charged and thus more acidic compared to a non-deamidated protein (see e.g. Robinson, Protein Deamidation , (2002) PNAS 99(8):5283-88). The percentage of acidic forms of a protein can be determined by ion exchange chromatography (eg, cation exchange high performance liquid chromatography) or imaging capillary isoelectric focusing (icIEF). In certain embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the multispecific binding proteins in the pharmaceutical formulation are one of the After the above stress, it is in the main charge configuration. In certain embodiments, no more than 10%, 20%, 30%, 40%, or 50% of the multispecific binding proteins in the pharmaceutical formulation are, after one or more stresses disclosed herein, It is in the acidic form.

Stability can also be measured by denaturing the protein with sodium dodecyl sulfate (SDS) and then measuring the purity of the multispecific binding protein by electrophoresis. A protein sample may be denatured in the presence or absence of an agent that reduces protein disulfide bonds (eg, β-mercaptoethanol). In certain embodiments, the purity of the multispecific binding protein in the pharmaceutical formulation, as measured by capillary electrophoresis after denaturing the protein sample under reducing conditions (e.g., in the presence of β-mercaptoethanol), is At least 95%, 96%, 97%, 98%, or 99% after one or more stresses disclosed herein. In certain embodiments, the purity of the multispecific binding protein in the pharmaceutical formulation, as measured by capillary electrophoresis after denaturing the protein sample under reducing conditions (e.g., in the presence of β-mercaptoethanol), is At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% after one or more stresses disclosed herein. In certain embodiments, the purity of the multispecific binding protein in the pharmaceutical formulation, as measured by capillary electrophoresis after denaturing the protein sample under non-reducing conditions, is one or more of at least 95%, 96%, 97%, 98%, or 99% after stress. In certain embodiments, the purity of the multispecific binding protein in the pharmaceutical formulation, as measured by capillary electrophoresis after denaturing the protein sample under reducing conditions, is one or more of the at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% after stress is.

Stability can also be measured by determining protein solution parameters by dynamic light scattering. Z-average and polydispersity index (PDI) values indicate the average diameter of particles in solution, and these measurements increase when aggregates are present in solution. The monomer %Pd values indicate the spread of different monomers detected, with lower values indicating preferred monodisperse solutions. The monomer size detected by DLS is useful to confirm that the predominant population is monomeric and to characterize higher order aggregates that may be present. In certain embodiments, the Z-mean value of a pharmaceutical formulation does not increase by more than 5%, 10%, or 15% after one or more stresses disclosed herein. In certain embodiments, the Z-mean value of a pharmaceutical formulation does not increase by more than 10%, 20%, 30%, 40%, or 50% after one or more stresses disclosed herein.

In addition to evaluating the above parameters, stability can be measured by determining functional properties of the protein. The multispecific binding proteins disclosed herein bind CLEC12A, NKG2D, and CD16. Thus, according to certain embodiments, CLEC12A (e.g., human CLEC12A) after one or more stresses disclosed herein as measured by dissociation constant (K _D ) or maximal binding response (capture level) is 1%, 2%, 3%, 4%, 5%, 10%, 20%, or not lower than 30%. In certain embodiments, multispecificity to one or more post-stress NKG2Ds (e.g., human NKG2D) disclosed herein as measured by dissociation constant (K _D ) or maximal binding response (capture level) The binding affinity of the sex binding protein was 1%, 2%, 3%, 4%, 5%, 10%, 20%, or 30% higher than that of the control sample (unstressed). Not too low. In certain embodiments, multispecificity to one or more post-stress CD16 (e.g., human CD16aV158) disclosed herein as measured by dissociation constant (K _D ) or maximal binding response (capture level) The binding affinity of the sex binding protein was 1%, 2%, 3%, 4%, 5%, 10%, 20%, or 30% higher than that of the control sample (unstressed). Not too low.

The multispecific binding proteins disclosed herein have the ability to mediate cytotoxicity against CLEC12A-expressing tumor cells by NK cells. In certain embodiments, a multispecific binding protein that mediates such cytotoxicity following one or more stresses disclosed herein as measured by the EC50 of the multispecific binding protein in a mixed culture cytotoxicity assay. The potency of the binding protein is no more than 1%, 2%, 3%, 4%, 5%, 10%, 20%, or 30% greater than that in control samples (unstressed). In certain embodiments, multispecific binding proteins that mediate such cytotoxicity following one or more stresses disclosed herein as measured by maximal lysis of target cells in a mixed culture cytotoxicity assay. is no more than 1%, 2%, 3%, 4%, 5%, 10%, 20%, or 30% greater than that in control samples (not exposed to stress).

Exemplary methods for determining the stability of multispecific binding proteins in pharmaceutical formulations are described in Examples 13 and 14 of this disclosure. Furthermore, the stability of a protein can be measured by the binding affinity of a multispecific binding protein for its target or multispecific binding in certain in vitro assays such as the NK cell activation assay and cytotoxicity assays described in WO2018/152518. It can be evaluated by measuring the biological activity of the protein.

Dosage Forms Pharmaceutical formulations may be prepared and stored as liquid formulations or in lyophilized form. In certain embodiments, the pharmaceutical formulation is a liquid formulation for storage at 2-8°C (eg, 4°C) or a frozen formulation for storage at -20°C or below. The sugar or sugar alcohol in the formulation is used as a cryoprotectant.

Prior to use, the pharmaceutical formulation may be diluted or reconstituted with an aqueous carrier appropriate to the route of administration. Other exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions such as phosphate buffered saline, sterile saline solution, Ringer's solution, or dextrose solution. is mentioned. For example, if a pharmaceutical formulation is prepared for intravenous administration, the pharmaceutical formulation may be diluted with a 0.9% sodium chloride (NaCl) solution. In certain embodiments, the diluted pharmaceutical formulations are isotonic and suitable for administration by intravenous infusion.

The pharmaceutical formulation contains a concentration of the multispecific binding protein suitable for storage. In certain embodiments, the pharmaceutical formulation contains 10-200 mg/mL, 10-150 mg/mL, 10-100 mg/mL, 10-50 mg/mL, 10-40 mg/mL, 10-30 mg/mL, 10-25 mg/mL /mL, 10-20 mg/mL, 10-15 mg/mL, 20-200 mg/mL, 20-150 mg/mL, 20-100 mg/mL, 20-50 mg/mL, 20-40 mg/mL, 20-30 mg/mL , 20-25 mg/mL, 50-200 mg/mL, 50-150 mg/mL, 50-100 mg/mL, 100-200 mg/mL, 100-150 mg/mL, or 150-200 mg/mL. Contains protein. In specific embodiments, the pharmaceutical formulation contains , 90 mg/mL, 100 mg/mL, 150 mg/mL, or 200 mg/mL of multispecific binding protein.

In certain embodiments, the pharmaceutical formulation is packaged in a container (eg, vial, bag, pen, or syringe). In certain embodiments, the formulation can be a lyophilized or liquid formulation. In certain embodiments, the amount of multispecific binding protein in the container is suitable for administration as a single dose. In certain embodiments, the amount of multispecific binding protein in the container is suitable for administration as multiple doses. In certain embodiments, the pharmaceutical formulation comprises the multispecific binding protein in an amount of 0.1-2000 mg. In certain embodiments, the pharmaceutical formulation contains 1-2000 mg, 10-2000 mg, 20-2000 mg, 50-2000 mg, 100-2000 mg, 200-2000 mg, 500-2000 mg, 1000-2000 mg, 0.1-1000 mg, 1 ~1000mg, 10~1000mg, 20~1000mg, 50~1000mg, 100~1000mg, 200~1000mg, 500~1000mg, 0.1~500mg, 1~500mg, 10~500mg, 20~500mg, 50~500mg, 100 ~500 mg, 200-500 mg, 0.1-200 mg, 1-200 mg, 10-200 mg, 20-200 mg , 50-100 mg, 0.1-50 mg, 1-50 mg, 10-50 mg, 20-50 mg, 0.1-20 mg, 1-20 mg, 10-20 mg, 0.1-10 mg, 1-10 mg, or 0.1-10 mg. Contains multispecific binding proteins in amounts of 1-1 mg. In certain embodiments, the pharmaceutical formulation contains 0.1 mg, 1 mg, 2 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 400 mg. , 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1500 mg, or 2000 mg of the multispecific binding protein.

IV. Therapeutic Applications This application provides methods for treating autoimmune diseases or cancer using the multispecific binding proteins described herein and/or the pharmaceutical compositions described herein (e.g., pharmaceutical formulation). This method can be used to treat various cancers that express CLEC12A.

Treatments can be characterized according to the cancer being treated. Cancers to be treated can be characterized according to the presence of particular antigens expressed on the surface of cancer cells.

Cancers characterized by expression of CLEC12A include, but are not limited to, solid tumor indications such as gastric, esophageal, lung, triple-negative breast, colorectal, and head and neck cancers.

The proteins, conjugates, cells, and/or pharmaceutical compositions of this disclosure can be used to treat a variety of cancers, including but not limited to cancers in which the cancer cells or cells within the cancer microenvironment express CLEC12A. can be

Treatments can be characterized according to the cancer being treated. For example, in certain embodiments, the cancer is acute myelogenous leukemia, multiple myeloma, diffuse large B-cell lymphoma, thymoma, adenocystic carcinoma, gastrointestinal cancer, renal cancer, breast cancer, glioblastoma , lung cancer, ovarian cancer, brain cancer, prostate cancer, pancreatic cancer, or melanoma. In some embodiments, the cancer is a hematologic malignancy or leukemia. In certain embodiments, the cancer is acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), myelodysplasia, myelodysplastic syndrome, acute T lymphoblastic leukemia, or acute promyelocytic chronic myelomonocytic leukemia, chronic myelomonocytic leukemia, or myeloblastic manifestations of chronic myelogenous leukemia.

In certain embodiments, the AML is minimal residual disease (MRD). In certain embodiments, the MRD comprises CLEC12A-ITD ((Fms-like tyrosine kinase 3)-intragenic tandem duplication (ITD)), NPM1 (nucleophosmin 1), DNMT3A (DNA methyltransferase gene DNMT3A), and IDH ( Characterized by the presence or absence of mutations selected from isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). In certain embodiments, MDS is MDS with multilineage dysplasia (MDS-MLD), MDS with single lineage dysplasia (MDS-SLD), MDS with ringed sideroblasts (MDS-RS ), MDS with blast proliferation (MDS-EB), MDS with isolated del (5q), and unclassified MDS (MDS-U). In certain embodiments, the MDS is a primary MDS or a secondary MDS.

In certain embodiments, ALL is selected from B-cell acute lymphoblastic leukemia (B-ALL) and T-cell acute lymphoblastic leukemia (T-ALL). In certain embodiments, the MPN is selected from polycythemia vera, essential thrombocythemia (ET), and myelofibrosis. In certain embodiments, the non-Hodgkin's lymphoma is selected from B-cell lymphoma and T-cell lymphoma. In certain embodiments, the lymphoma is chronic lymphocytic leukemia (CLL), lymphoblastic lymphoma (LPL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), primary mediastinal large cell From type B-cell lymphoma (PMBL), follicular lymphoma, mantle cell lymphoma, pilocytic leukemia, plasma cell myeloma (PCM) or multiple myeloma (MM), mature T/NK cell tumors, and histiocytic tumors selected.

In certain embodiments, the cancer is a solid tumor. In certain other embodiments, the cancer is brain cancer, bladder cancer, breast cancer, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, esophageal cancer, leukemia, lung cancer, liver cancer, melanoma, ovarian cancer , pancreatic, prostate, rectal, renal, gastric, testicular, or uterine cancer. In yet other embodiments, the cancer is angiogenic tumor, squamous cell carcinoma, adenocarcinoma, small cell carcinoma, melanoma, glioma, neuroblastoma, sarcoma (eg, angiosarcoma or chondrosarcoma), laryngeal Cancer, parotid cancer, biliary tract cancer, thyroid cancer, acral lentiginous melanoma, actinic keratosis, acute lymphocytic leukemia, acute myeloid leukemia, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma , anal canal cancer, anal cancer, anorectal cancer, astrocytic tumor, Bartholin adenocarcinoma, basal cell carcinoma, cholangiocarcinoma, bone cancer, bone marrow cancer, bronchial carcinoma, bronchial adenocarcinoma, carcinoid, cholangiocarcinoma, chondrosarcoma, choroid Plexus papilloma/choroid plexus carcinoma, chronic lymphocytic leukemia, chronic myeloid leukemia, clear cell carcinoma, connective tissue carcinoma, cystadenoma, gastrointestinal cancer, duodenal cancer, endocrine cancer, endoderm sinus tumor, endometrium Hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, endothelial cell carcinoma, ependymal carcinoma, epithelial cell carcinoma, Ewing sarcoma, eye and orbital cancer, female genital cancer, focal nodular hyperplasia, gallbladder cancer, stomach Sinus cancer, gastric fundus carcinoma, gastrinoma, glioblastoma, glucagonoma, heart cancer, hemangioblastoma, hemangioendothelioma, hemangioma, liver adenoma adenomatosis, hepatobiliary cancer, hepatocellular carcinoma, Hodgkin's disease, ileal cancer , insulinoma, intraepithelial neoplasia, intraepithelial squamous cell tumor, intrahepatic cholangiocarcinoma, invasive squamous cell carcinoma, jejunal cancer, joint cancer, Kaposi's sarcoma, pelvic cancer, large cell carcinoma, colon cancer, leiomyosarcoma, malignant lentigo melanoma, lymphoma, male genital cancer, malignant melanoma, malignant mesothelial tumor, medulloblastoma, medulloepithelioma, meningeal carcinoma, mesothelial carcinoma, metastatic carcinoma, mouth cancer, mucoepidermoid cancer, multiple myeloma, muscle cancer, nasal tract cancer, nervous system cancer, neuroepithelial adenocarcinoma nodular melanoma, non-epithelial skin cancer, non-Hodgkin's lymphoma, oat cell carcinoma, oligodendroglial carcinoma, oral cancer cavity cancer), osteosarcoma, papillary serous adenocarcinoma, penile cancer, pharyngeal cancer, pituitary tumor, plasmacytoma, pseudosarcoma, pulmonary blastoma, rectal cancer, renal cell carcinoma, respiratory system cancer, retinoblast tumor, rhabdomyosarcoma, sarcoma, serous carcinoma, sinus carcinoma, skin cancer, small cell carcinoma, small bowel cancer, smooth muscle carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, spinal cancer, squamous cell carcinoma, striatal muscle Cancer, submesothelial carcinoma, superficial metastatic melanoma, T-cell leukemia, tongue cancer, undifferentiated cancer, ureteral cancer, urethral cancer, bladder cancer, urinary tract cancer, cervical cancer, endometrial cancer, uterine melanoma carcinoma, vaginal cancer, wart cancer, VIPoma, vulvar cancer, well-differentiated carcinoma, or Wilms tumor.

In certain other embodiments, the cancer is non-Hodgkin's lymphoma, such as B-cell lymphoma or T-cell lymphoma. In certain embodiments, the non-Hodgkin's lymphoma is B-cell lymphoma, e.g., diffuse large B-cell lymphoma, primary mediastinal B-cell lymphoma, follicular lymphoma, small lymphocytic lymphoma, mantle cell lymphoma, marginal Zone B-cell lymphoma, extranodal marginal zone B-cell lymphoma, nodal marginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia, or primary It is a central nervous system (CNS) lymphoma. In certain other embodiments, the non-Hodgkin's lymphoma is T-cell lymphoma, e.g., precursor T-lymphoblastic lymphoma, peripheral T-cell lymphoma, cutaneous T-cell lymphoma, angioimmunoblastic T-cell lymphoma, extranodal natural Killer/T-cell lymphoma, enteropathic T-cell lymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplastic large cell lymphoma, or peripheral T-cell lymphoma.

IV. Combination Therapy Another aspect of the present application provides combination therapy. The multispecific binding proteins described herein can be used in combination with additional therapeutic agents to treat autoimmune diseases or to treat cancer.

Exemplary therapeutic agents that may be used as part of combination therapy in treating autoimmune inflammatory diseases are described in Li et al. (2017) Front. Pharmacol. 8:460, which include, for example, non-steroidal anti-inflammatory drugs (NSAIDs) (eg, COX-2 inhibitors), glucocorticoids (eg, prednisone/prednisolone, methylprednisolone, and fluorinated glucocorticoids, such as dexamethasone and betamethasone), disease-modifying antirheumatic drugs (DMARDs), such as methotrexate, leflunomide, gold compounds, sulfasalazine, azathioprine, cyclophosphamide, antimalarials, D-penicillamine, and cyclosporine ), TNF-rich biologics (e.g., infliximab, etanercept, adalimumab, golimumab, certolizumab pegol, and their biosimilars), and other biologics that target CTLA-4 (e.g., abatacept), IL-6 receptors (eg tocilizumab), IL-1 (eg anakinra), Th1 immune response (IL-12/IL-23) (eg ustekinumab), Th17 immune response (IL-17) ( secukinumab) and CD20 (eg rituximab).

Exemplary therapeutic agents that can be used as part of a combination therapy in the treatment of cancer include, for example, radiation, mitomycin, tretinoin, ribomustine, gemcitabine, vincristine, etoposide, cladribine, mitobronitol, methotrexate, doxorubicin, carboquone, pentostatin. , nitracrine, dinostatin, cetrorelix, letrozole, raltitrexed, daunorubicin, fadrozole, fotemustine, thymalfasine, sobuzoxan, nedaplatin, cytarabine, bicalutamide, vinorelbine, vesnarinone, aminoglutethimide, amsacrine, proglumide, elliptinium acetate, ketanserin, doxfluridine, Etretinate, isotretinoin, streptozocin, nimustine, vindesine, flutamide, drogenil, butocine, carmofur, lazoxan, schizofiran, carboplatin, mitractol, tegafur, ifosfamide, prednimustine, picivanil, levamisole, teniposide, improsulfan, enocitabine, lisuride, oxymetholone , tamoxifen, progesterone, mepitiostane, epithiostanol, formestane, interferon-alpha, interferon-2-alpha, interferon-beta, interferon-gamma (IFN-γ), colony-stimulating factor-1, colony-stimulating factor-2, denileukin diffitox , interleukin-2, luteinizing hormone-releasing factor, and variations of the foregoing agents that may exhibit differential binding to their cognate receptors, or increased or decreased serum half-lives.

An additional class of drugs that may be used as part of combination therapy in the treatment of cancer are immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include (i) cytotoxic T lymphocyte-associated antigen 4 (CTLA4), (ii) programmed cell death protein 1 (PD1), (iii) PDL1, (iv) LAG3, (v ) B7-H3, (vi) B7-H4, and (vii) TIM3. The CTLA4 inhibitor ipilimumab is approved by the US Food and Drug Administration for the treatment of melanoma.

Still other agents that can be used as part of combination therapy in the treatment of cancer are monoclonal antibody agents that target non-checkpoint targets (e.g. Herceptin) and non-cytotoxic agents (e.g. tyrosine kinase inhibitors). be.

Still other categories of anticancer agents include, for example, (i) ALK inhibitors, ATR inhibitors, A2A antagonists, base excision repair inhibitors, Bcr-Abl tyrosine kinase inhibitors, Bruton's tyrosine kinase inhibitors, CDC7 inhibitors. , CHK1 inhibitors, cyclin-dependent kinase inhibitors, DNA-PK inhibitors, inhibitors of both DNA-PK and mTOR, DNMT1 inhibitors, DNMT1 inhibitors plus 2-chlorodeoxyadenosine, HDAC inhibitors, hedges Hogg signaling pathway inhibitors, IDO inhibitors, JAK inhibitors, mTOR inhibitors, MEK inhibitors, MELK inhibitors, MTH1 inhibitors, PARP inhibitors, phosphoinositide 3-kinase inhibitors, inhibitors of both PARP1 and DHODH , proteasome inhibitors, topoisomerase II inhibitors, tyrosine kinase inhibitors, VEGFR inhibitors, and WEE1 inhibitors; (ii) OX40, CD137, CD40, GITR, CD27, HVEM, TNFRSF25, or ICOS and (iii) cytokines selected from IL-12, IL-15, GM-CSF, and G-CSF.

Proteins of the present application may also be used as an adjunct to surgical removal of primary tumors.

The amounts of multispecific binding protein and additional therapeutic agent, as well as the relative timings of administration, can be selected to achieve the desired combined therapeutic effect. For example, when administering combination therapy to a patient in need of such administration, the combination therapeutic agents, or pharmaceutical composition(s) comprising the therapeutic agents, may be administered in any order, e.g. , together, at the same time, and the like. Further, for example, the multispecific binding protein may be administered during the time that the additional therapeutic agent(s) exerts its prophylactic or therapeutic effect, or vice versa.

V. Pharmaceutical Compositions The present disclosure also features pharmaceutical compositions that include a therapeutically effective amount of a protein described herein. This composition may be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers may also be included in the composition for proper formulation. Suitable formulations for use in the present disclosure are available from Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa.; , 17th ed. , 1985. For a brief review of methods for drug delivery see, eg, Langer (Science 249:1527-1533, 1990).

Intravenous drug delivery formulations of the present disclosure may be contained in a bag, pen, or syringe. In certain embodiments, the bag may be connected to channels containing tubes and/or needles. In certain embodiments, the formulation may be a lyophilized formulation or a liquid formulation. In certain embodiments, the formulation may be freeze-dried (lyophilized) and contained in about 12-60 vials. In certain embodiments, the formulation is lyophilized and 45 mg of lyophilized formulation may be contained in one vial. In certain embodiments, about 40 mg to about 100 mg of lyophilized formulation may be contained in one vial. In certain embodiments, lyophilized formulations from 12, 27, or 45 vials are combined to obtain a therapeutic dose of protein in an intravenous drug formulation. In certain embodiments, the formulation may be a liquid formulation and may be stored as from about 250 mg/vial to about 1000 mg/vial. In certain embodiments, the formulation can be a liquid formulation and can be stored as about 600 mg/vial. In certain embodiments, the formulation may be a liquid formulation and stored as about 250 mg/vial.

The protein may be present in a liquid aqueous formulation comprising a therapeutically effective amount of the protein in a buffer forming the formulation.

These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or can be lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparation is typically between 3 and 11, more preferably between 5 and 9 or between 6 and 8, most preferably between 7 and 8, such as 7 and 7.5. is. A composition presented in solid form may be packaged in a plurality of single dose units each containing a fixed amount of the above agent(s). Solid form compositions may also be packaged in containers for plasticity.

In certain embodiments, the present disclosure provides mannitol, citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, sodium dihydrogen phosphate dihydrate, sodium chloride, polysorbate 80, water, and sodium hydroxide to provide formulations with extended shelf life comprising the proteins of the present disclosure.

In certain embodiments, an aqueous formulation is prepared comprising a protein of the disclosure in a pH buffer. The buffer may have a pH ranging from about 4 to about 8, such as from about 4.5 to about 6.0, or from about 4.8 to about 5.5; It may have a pH from 0 to about 5.2. Ranges intermediate to the above pHs are also intended to be part of this disclosure. For example, ranges of values using combinations of any of the above values as upper and/or lower limits are intended to be included. Examples of buffers that control pH within this range include acetates (e.g. sodium acetate), succinates (e.g. sodium succinate), gluconates, histidine, citrates and other organic acids. Buffers are included.

In certain embodiments, the formulation comprises a buffer system comprising citrate and phosphate to maintain pH in the range of about 4 to about 8. In certain embodiments, the pH range may be from about 4.5 to about 6.0, or from about pH 4.8 to about pH 5.5, or from about 5.0 to about 5.2. In certain embodiments, the buffer system includes citric acid monohydrate, sodium citrate, disodium phosphate dihydrate, and/or sodium dihydrogen phosphate dihydrate. In certain embodiments, the buffer system comprises about 1.3 mg/mL citric acid (e.g., 1.305 mg/mL), about 0.3 mg/mL sodium citrate (e.g., 0.305 mg/mL), About 1.5 mg/mL disodium phosphate dihydrate (e.g., 1.53 mg/mL), about 0.9 mg/mL sodium dihydrogen phosphate dihydrate (e.g., 0.86 mg/mL ), and about 6.2 mg/mL sodium chloride (eg, 6.165 mg/mL). In certain embodiments, the buffer system comprises about 1 to about 1.5 mg/mL citric acid, about 0.25 to about 0.5 mg/mL sodium citrate, about 1.25 to about 1.75 mg/mL mL disodium phosphate dihydrate, about 0.7 to about 1.1 mg/mL sodium dihydrogen phosphate dihydrate, and about 6.0 to about 6.4 mg/mL sodium chloride . In certain embodiments, the pH of the formulation is adjusted with sodium hydroxide.

Polyols, which can act as tonicity agents and stabilize antibodies, may also be included in the formulation. Polyols are added to the formulation in amounts that may vary with respect to the desired isotonicity of the formulation. In certain embodiments, aqueous formulations may be isotonic. The amount of polyol added may also vary with respect to the molecular weight of the polyol. For example, smaller amounts of monosaccharides (eg, mannitol) may be added compared to disaccharides (such as trehalose). In certain embodiments, a polyol that can be used in the formulation as a tonicity agent is mannitol. In certain embodiments, the concentration of mannitol can be from about 5 to about 20 mg/mL. In certain embodiments, the concentration of mannitol can be from about 7.5 to about 15 mg/mL. In certain embodiments, the concentration of mannitol can be from about 10 to about 14 mg/mL. In certain embodiments, the concentration of mannitol can be about 12 mg/mL. In certain embodiments, polyol sorbitol may be included in the formulation.

A surfactant or surfactant may also be added to the formulation. Exemplary surfactants include nonionic surfactants such as polysorbates (eg, polysorbate 20, 80, etc.) or poloxamers (eg, poloxamer 188, etc.). The amount of surfactant added is such that it reduces aggregation of the formulated antibody and/or minimizes the formation of microparticles in the formulation and/or reduces adsorption. . In certain embodiments, the formulation may include a surfactant that is a polysorbate. In certain embodiments, the formulation may include the surfactant polysorbate 80 or Tween®80. Tween® 80 is a term used to describe polyoxyethylene (20) sorbitan monooleate (see Fiedler, Lexikon der Hifsstoffe, Editio Cantor Verlag Aulendorf, 4th ed., 1996). matter). In certain embodiments, formulations may contain from about 0.1 mg/mL to about 10 mg/mL polysorbate 80, or from about 0.5 mg/mL to about 5 mg/mL. In certain embodiments, about 0.1% polysorbate 80 may be added to the formulation.

In embodiments, the protein products of this disclosure are formulated as liquid formulations. Liquid formulations may be supplied at a concentration of 10 mg/mL in either USP/Ph Eur Type I50R vials, closed with rubber stoppers and sealed with aluminum crimp seal closures. The stopper may be made of USP and Ph Eur compliant elastomers. In certain embodiments, a vial can be filled with 61.2 mL of protein product solution to allow for an extractable volume of 60 mL. In certain embodiments, liquid formulations may be diluted with 0.9% saline.

In certain embodiments, liquid formulations of the present disclosure may be prepared as a solution at a concentration of 10 mg/mL in combination with stabilizing levels of sugar. In certain embodiments, liquid formulations may be prepared in an aqueous carrier. In certain embodiments, stabilizers may be added in amounts up to and including amounts that may result in undesirable or inappropriate viscosity for intravenous administration. In certain embodiments, the sugar can be a disaccharide, such as sucrose. In certain embodiments, liquid formulations may also include one or more of buffers, surfactants, and preservatives.

In certain embodiments, the pH of liquid formulations may be set by the addition of pharmaceutically acceptable acids and/or bases. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the base can be sodium hydroxide.

In addition to aggregation, deamidation is a common product variant of peptides and proteins that can occur during fermentation, harvest/cell clarification, purification, drug/drug product storage and sample analysis. Deamidation is the loss of _NH3 from a protein forming a succinimide intermediate that can undergo hydrolysis. A succinimide intermediate results in a 17 dalton mass loss of the parent peptide. Subsequent hydrolysis increases the mass by 18 Daltons. Isolation of the succinimide intermediate is difficult due to its instability under aqueous conditions. Therefore, deamidation is usually detectable as a mass increase of 1 Dalton. Deamidation of asparagine yields either aspartic acid or isoaspartic acid. Parameters affecting the rate of deamidation include pH, temperature, solvent permittivity, ionic strength, primary sequence, local polypeptide conformation and tertiary structure. Amino acid residues flanking Asn in the peptide chain influence the rate of deamidation. Gly and Ser following Asn in the protein sequence make it more susceptible to deamidation.

In certain embodiments, liquid formulations of the present disclosure may be stored under conditions of pH and humidity to prevent deamination of protein products.

Aqueous carriers of interest herein are those that are pharmaceutically acceptable (safe and non-toxic for human administration) and useful in the preparation of liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (eg phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution. .

Preservatives may optionally be added to the formulations herein to reduce bacterial action. The addition of preservatives, for example, can facilitate the production of multiple use (multidose) formulations.

Intravenous (IV) formulations may be the preferred route of administration in certain cases, such as when a patient is hospitalized after transplant with all drugs administered via the IV route. In certain embodiments, liquid formulations are diluted with 0.9% sodium chloride solution prior to administration. In certain embodiments, the diluted drug product for injection is isotonic and suitable for administration by intravenous infusion.

In certain embodiments, salts or buffer components may be added in amounts of 10 mM to 200 mM. Salts and/or buffers are pharmaceutically acceptable and are derived from a variety of known acids (inorganic and organic), including "base-forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate, carbonate, citrate buffer, in which case sodium, potassium or ammonium ions may serve as counterions.

Preservatives may optionally be added to the formulations herein to reduce bacterial action. Addition of preservatives, for example, can facilitate the production of multiple use (multidose) formulations.

Aqueous carriers of interest herein are those that are pharmaceutically acceptable (safe and non-toxic for human administration) and useful in the preparation of liquid formulations. Exemplary carriers include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (eg, phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution. be done.

A protein of the disclosure may be present in a lyophilized formulation comprising the protein and a cryoprotectant. A cryoprotectant may be a sugar, eg, a disaccharide. In certain embodiments, the cryoprotectant may be sucrose or maltose. The lyophilized formulation may also include one or more of buffers, surfactants, bulking agents and/or preservatives.

A useful amount of sucrose or maltose for stabilizing a lyophilized pharmaceutical product may be a weight ratio of protein to sucrose or maltose of at least 1:2. In certain embodiments, the weight ratio of protein to sucrose or maltose can be from 1:2 to 1:5.

In certain embodiments, the pH of the formulation may be set by the addition of pharmaceutically acceptable acids and/or bases prior to lyophilization. In certain embodiments, the pharmaceutically acceptable acid may be hydrochloric acid. In certain embodiments, the pharmaceutically acceptable base may be sodium hydroxide.

Prior to lyophilization, the pH of the solution containing the protein of the disclosure can be adjusted between 6 and 8. In certain embodiments, the pH range of the lyophilized pharmaceutical may be between 7-8.

In certain embodiments, salts or buffer components may be added in amounts of 10 mM to 200 mM. Salts and/or buffers are pharmaceutically acceptable and derived from a variety of known acids (inorganic and organic), including "base-forming" metals or amines. In certain embodiments, the buffer may be a phosphate buffer. In certain embodiments, the buffer may be a glycinate, carbonate, citrate buffer, in which case sodium, potassium or ammonium ions may serve as counterions.

In certain embodiments, "bulking agents" may be added. A "bulking agent" is one that adds mass to the lyophilized mixture and contributes to the physical structure of the lyophilized cake (e.g., facilitates production of an essentially uniform lyophilized cake that maintains an open pore structure). is a compound. Exemplary bulking agents include mannitol, glycine, polyethylene glycol, and sorbitol. Lyophilized formulations of polyspecific binding proteins described in this application may include such bulking agents.

Preservatives may optionally be added to the formulations herein to reduce bacterial action. Addition of preservatives, for example, can facilitate the production of multiple use (multidose) formulations.

In certain embodiments, a lyophilized pharmaceutical product may be composed of an aqueous carrier. Aqueous carriers of interest herein are those that are pharmaceutically acceptable (safe and non-toxic for human administration) and useful for the preparation of liquid formulations after lyophilization. Exemplary diluents include sterile water for injection (SWFI), bacteriostatic water for injection (BWFI), pH buffered solutions (eg, phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution. be done.

In certain embodiments, the lyophilized pharmaceutical product of the present disclosure is reconstituted with either Sterile Water for Injection, USP (SWFI) or 0.9% Sodium Chloride Injection, USP. During reconstitution, the lyophilized powder dissolves into solution.

In certain embodiments, the lyophilized protein product of the present disclosure is made up of about 4.5 mL of water for injection and diluted with 0.9% saline (sodium chloride solution).

The actual dosage level of the active ingredients in the pharmaceutical compositions of the multispecific binding proteins described in this application will be as desired for the particular patient, composition and mode of administration, without being toxic to the patient. Variations may be made to obtain an amount of active ingredient effective to achieve the therapeutic response.

A particular dose may be a flat dose for each patient, eg, 50-5000 mg of protein. Alternatively, a patient's dose may be adjusted to approximate the patient's body weight or surface area. Other factors in determining the appropriate dosage can include the disease or condition to be treated or prevented, severity of the disease, route of administration, and age, sex, and medical condition of the patient. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those skilled in the art, especially in light of the dosage information and assays disclosed herein. Dosages can also be determined by using known assays for determining dosages used in conjunction with appropriate dose-response data. Dosages for individual patients may be adjusted while monitoring disease progression. Blood levels of the targetable construct or conjugate in the patient can be measured to determine whether the dosage needs to be adjusted to reach or maintain effective concentrations. Pharmacogenomics can be used to determine which targetable constructs and/or conjugates and dosages thereof are likely to be most effective in a given individual (Schmitz et al., Clinica Chimica Acta 308:43-53, 2001; Steimer et al., Clinica Chimica Acta 308:33-41, 2001).

Generally, dosages based on body weight range from about 0.01 μg to about 100 mg per kg of body weight, such as from about 0.01 μg to about 100 mg/kg of body weight, from about 0.01 μg to about 50 mg/kg of body weight, about 0 .01 μg to about 10 mg/kg body weight, about 0.01 μg to about 1 mg/kg body weight, about 0.01 μg to about 100 μg/kg body weight, about 0.01 μg to about 50 μg/kg body weight, about 0.01 μg to about 10 μg/kg (body weight), about 0.01 μg to about 1 μg/kg (body weight), about 0.01 μg to about 0.1 μg/kg (body weight), about 0.1 μg to about 100 mg/kg (body weight), about 0.1 μg to about 50 mg/kg (body weight), about 0.1 μg to about 10 mg/kg (body weight), about 0.1 μg to about 1 mg/kg (body weight), about 0.1 μg to about 100 μg /kg body weight, about 0.1 μg to about 10 μg/kg body weight, about 0.1 μg to about 1 μg/kg body weight, about 1 μg to about 100 mg/kg body weight, about 1 μg to about 50 mg/kg (body weight), about 1 μg to about 10 mg/kg (body weight), about 1 μg to about 1 mg/kg (body weight), about 1 μg to about 100 μg/kg (body weight), about 1 μg to about 50 μg/kg (body weight), about 1 μg to about 10 μg/kg (body weight), about 10 μg to about 100 mg/kg (body weight), about 10 μg to about 50 mg/kg (body weight), about 10 μg to about 10 mg/kg (body weight), about 10 μg to about 1 mg/kg ( body weight), about 10 μg to about 100 μg/kg (body weight), about 10 μg to about 50 μg/kg (body weight), about 50 μg to about 100 mg/kg (body weight), about 50 μg to about 50 mg/kg (body weight), about 50 μg to About 10 mg/kg (body weight), about 50 μg to about 1 mg/kg (body weight), about 50 μg to about 100 μg/kg (body weight), about 100 μg to about 100 mg/kg (body weight), about 100 μg to about 50 mg/kg (body weight) ), about 100 μg to about 10 mg/kg (body weight), about 100 μg to about 1 mg/kg (body weight), about 1 mg to about 100 mg/kg (body weight), about 1 mg to about 50 mg/kg (body weight), about 1 mg to about 10 mg/kg body weight, about 10 mg to about 100 mg/kg body weight, about 10 mg to about 50 mg/kg body weight, about 50 mg to about 100 mg/kg body weight.

Doses may be given once or more daily, weekly, monthly or yearly, or even once every 2-20 years. Persons of ordinary skill in the art can readily estimate repetition rates for dosing based on measured residence times and concentrations of the targetable construct or complex in bodily fluids or tissues. Administration of the multispecific binding proteins described in this application can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, intracavitary, by perfusion through a catheter, or by direct intralesional injection. may be by It may be administered more than once a day, more than once a week, more than once a month, and more than once a year.

The above description provides multiple aspects and embodiments of the multispecific binding proteins described in this application. This patent application expressly contemplates all combinations and permutations of aspects and embodiments. The use of any and all examples, or exemplary language herein, e.g., "including" or "including," is merely intended to better describe the multispecific binding proteins described in this application. , do not pose limitations on the scope of the disclosure unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the multispecific binding proteins described in this application.

The following examples are merely illustrative and are in no way intended to limit the scope or content of the multispecific binding proteins described in this application.

Example 1. Characterization of Supernatants of Selected Hybridoma Clones CLEC12A-specific antibodies were generated by immunizing BALB/c mice with the hCLEC12A-His fusion protein. Supernatants from 16 hybridomas were assessed for CLEC12A binding by Bio-layer Interferometry (BLI) binding using OctetRed384 (ForteBio). These 16 hybridomas were further analyzed for binding to human and cynomolgus monkey CLEC12A expressed on the cell surface of isogenic RMA cells; no binding to cynomolgus monkey CLEC12A was observed. The estimated kinetic parameters are shown in Table 11 and the binding traces are shown in FIG. Nine clones were selected for further study. The ability of these nine clones to bind hCLEC12A-His RMA and CLEC12A-expressing cancer cell lines U937 and PL21 was further analyzed by high-resolution surface plasmon resonance (SPR). This experiment was performed at 37° C. to mimic physiological temperature using a Biacore 8K instrument.

Binning of hybridoma fusions relative to reference mAbs was performed by BLI using OctetRed384 (ForteBio). Briefly, hybridoma supernatants were loaded onto anti-mouse IgG capture sensor chips for 15 minutes and equilibrated with PBSF for 5 minutes. Sensors were soaked in 200 nM hCLEC12A-His and allowed to bind for 180 seconds before soaking in 100 nM reference CLEC12A mAb. An increase in response units indicated that the hybridoma did not compete with the reference mAb, while no increase in signal indicated that the hybridoma competed with the reference mAb. The VH and VL sequences of these reference antibodies are shown in Table 12.

By binning analysis, five of the hybridomas, namely 12F8. G3, 13E01, 15A10. G8, 20D6. A8, and 23A5. It was demonstrated that the antibody generated from D4 did not compete with the reference antibody for binding to hCLEC12A-His. Hybridoma binding to isogenic human CLEC12A (hCLEC12A) and cross-reactivity with cynomolgus monkey CLEC12A (cCLEC12A) also measure antibody binding to isogenic RMA cells expressing CLEC12A and the U937AML cancer cell line. It was evaluated by

Briefly, RMA cells were transduced with retroviral vectors encoding cCLEC12A or hCLEC12A. Binding of α-CLEC12A mAb from crude hybridoma harvests to hCLEC12A or cCLEC12A isogenic cell lines and to CLEC12A+U937 (ATCC Catalog No. CRL-1593.2) cancer cell lines was performed as follows. 100,000 RMA, REH, or SEM cells were added per well of a 96-well round bottom plate. Cells were spun down and the pellet was gently dissociated by vortexing. 100 μL of Zombie live/dead dye (PBS+1:2000 dye) was added per well and incubated for 20 minutes at room temperature in the dark. Cells were washed with 200 μL of FACS buffer (PBS+2% FBS)). 50 μL of hybridoma supernatant was added to the washed cells and the mixture was incubated on ice in the dark for 30 minutes. After washing the cells twice with FACS buffer, 50 μL of anti-mouse Fc-PE secondary reagent (1:200 dilution) was added and incubated on ice in the dark for 20 minutes. After incubation, cells were washed and then fixed with 50 μL of 4% paraformaldehyde for 15 minutes on ice. Fixed cells were washed with FACS buffer, resuspended in 200 μL FACS buffer and stored at 4° C. until ready for acquisition. Samples of cells resuspended in FACS buffer were run on a BD FACSCelesta with HTS (High Throughput Sampler) to measure binding affinity of antibodies to isogenic RMA cells expressing CLEC12A and U937AML cancer cell lines. bottom.

The binding affinity of the hybridoma supernatants to PL21 AML cancer cells (DSMZ catalog number ACC536), a human AML cell line reported to express CLEC12A, was also measured. As shown in Table 11, 9 clones showed binding affinity to cancer cells expressing hCLEC12A. No binding to cCLEC12A was observed.
Example 2. Analysis of purified anti-CLEC12A mouse antibodies

In this example, kinetic parameters and binding affinities of purified anti-CLEC12A mouse antibodies were analyzed. Based on the analysis described in Example 1, eight hybridomas (9F11.B7, 12F8.G3, 16B8.C8, 15A10.G8, 20D6.A8, 9E4.B7, 13E1.A4, and 23A5.D4) were Selected for subcloning and sequencing. Each subclone was purified from hybridoma culture and binding to hCLEC12A-His and cCLEC12A-His was assessed by SPR as shown in FIG. Data from these experiments are shown in FIG. 19A. Antibody 9E4. B7, 9F11. B7, 12F8. G3, and 16B8. C8 bound only to hCLEC12A (Fig. 19A); whereas antibody 13El.C8 bound only to hCLEC12A (Figure 19A); A4, 15 A10. G8, 20D6. A8, and 23A5. D4 bound to both hCLEC12A and cCLEC12A (Fig. 19B). Kinetic constants and binding affinities of hCLEC12A and cCLEC12A to purified murine subcloned mAbs are shown in Table 13.

The ability of the 8 purified subclonal mAbs to bind to CLEC12A-expressing cells was assessed by FACS analysis using the human CLEC12A-positive PL21 cancer cell line. As shown in FIG. 20, 9E4. B7, 9F11. B7 and 16B8. C8 all bound to the PL21 cell line with sub-nanomolar EC50 values; B7 showed low affinity heterogeneous binding to recombinant hCLEC12A-His, thus 9F11. B7 and 16B8. Only C8 met the criteria for both recombinant protein binding and cell binding (Table 14). 15A10. G8, 13E1. A4, 20D6. A8, and 23A5. Although D4 showed binding to recombinant human and cyno (cyno) CLEC12A in SPR assays, these mAbs failed to recognize cancer cells and the binding epitope between recombinant and cell surface-expressed CLEC12A This suggests a difference in the higher-order structure of As shown, both Merus-CLL1 and Genentech-h6E7 mAbs bound PL21 with significantly inferior EC50 values compared to the novel CLEC12A hybridoma clone.

To assess whether the mAb binds to CLEC12A in a glycosylation-independent manner, clone 16B8. C8 and 9F11. Binding of B7 was assessed by SPR (Figure 21). As shown in the sensorgrams and Table 15, 16B8. C8 and 9F11. Both B7 bind to desialylated and deglycosylated versions of hCLEC12A without loss of affinity, suggesting that antibody interaction with hCLEC12A is not affected by the glycosylation status of the target. ing.

Example 3. Putative Sequence Liability Analysis 16B8. C8 and 9F11. The CDRs of the B7 antibody (identified by Chothia) were examined for potential sequence liability. We considered the following potential liabilities. M (potential oxidation sites); NG, NS and NT sequence motifs (potential deamidation sites); DG, DS and DT sequence motifs (potential isomerization sites); DP sequence motifs (potential chemical site of hydrolysis). Results are summarized in Table 16.

Variants of these antibodies were designed to remove putative sequence liability motifs.

Example 4. Humanization of Subclones Based on the data collected on the kinetics and affinity of recombinant hCLEC12A protein, binding to cell surface expressed hCLEC12A, and binding to AML cancer cell lines, two murine hybridoma subclones, namely 16B8. . C8 and 9F11. B7 was selected for humanization.

16B8. The C8 antibody was humanized. Back mutations were introduced in the framework regions to generate variants with the VH and VL sequences of scFv-1292 to scFv-1309 and scFv-1602 and scFv-2061.

9F11. The B7 antibody was humanized. Back mutations were introduced in the framework regions to create variants AB0186-AB0196.

Example 5. Selection of CLEC12A TriNKETs The following criteria were applied in selecting the best TriNKET candidates and the F3'-1602 TriNKET was selected as the lead candidate.
- Potent enhancement of NK activity against AML cancer cells - Higher potency than corresponding monoclonal antibodies - Binding to multiple AML cell lines - High affinity for recombinant hCLEC12A - Low affinity for human NKG2D - Cynomolgus monkey (cyno ) cross-reactivity with target - good developability profile:
Thermal stability of scFv above −65° C. Isoelectric point (pI) between −8 and 9
- Hydrophobic Interaction Chromatography (HIC) retention times similar to well-behaved therapeutic mAbs.
- High specificity for human CLEC12A - Clean polyspecific reagent (PSR) profile - Transient expression titers similar to mAb

Clone 16B8. All 18 humanized scFv variants of C8 were combined with the A49MI NKG2D Fab arms to create the humanized F3'CLEC12A TriNKET. The binding affinities of 18 semi-purified TriNKETs to hCLEC12A were assessed by screening SPR. The binding signals of the eight F3'-TriNKETs were less than 5 RU (approximately 15% of the expected signal performed in this assay), thus these TriNKETs were considered non-binders to hCLEC12A. The remaining 10 F3'-TriNKETs bound hCLEC12A with an affinity of less than 10 nM, as shown in Figure 22 and Table 17. However, potential N-glycosylation sites were inadvertently introduced into 8 of the 10 F3'-TriNKETs by introducing the mouse backmutation N at position H85. Only constructs F3′-1295 and F3′-1304 (containing an A at position H85) showed no N-glycosylation sequence liability; therefore, only these two constructs were carried forward for further characterization. .

F3'-1295 and F3'-1304 TriNKETs were tested for isogenic binding to hCLEC12A+RMA cells and using the methods described in Examples 5 or 6, as shown in FIG. The binding of F3'-1295 to cell surface expressed hCLEC12A was confirmed by the binding of hcFAE-A49. The binding of F3'-1304 to cell surface-expressed hCLEC12A was comparable to the CLL1-Merus control TriNKET, while hcFAE-A49. It was inferior compared to the CLL1-Merus control polyspecific binding protein.

The potency of F3′-1295 was evaluated in a primary NK cell-mediated cytotoxicity assay using HL-60 AML cells and using the method described in Example 8, as shown in FIG. F3'-1295 induced potent lysis of HL60 AML cells. The cell killing EC50 was similar and the maximal percentage of cell lysis was higher than the control hcFAE-A49. higher than CLL1-Merus TriNKET (Table 18).

Thermal stability of F3'-1295 was assessed by differential scanning calorimetry (DSC). Briefly, TriNKET was diluted to 0.5 mg/mL in PBS to perform DSC. 325 μL was added to a 96-well deep well plate along with matching buffer blanks. Thermograms were generated using a MicroCal PEAQ DSC (Malvern, Pa.). The temperature was increased from 20 to 100°C at 90°C/hour. Raw thermograms were background subtracted, a baseline model was splined, and a non-two state model was used to fit the data.

The melting temperature (T _m1 ) of the first transition of F3′-1295 was 60.9° C., 4° C. below the predefined criterion of ≥65° C. for the T _m of the first TriNKET transition. During humanization, the introduction of murine residues into the human framework (backmutation) may have affected thermostability. Reverting the backmutation to a human residue may therefore improve the thermostability of F3'-1295. Therefore, as shown in Table 19, the thermal stability of the 10 F3' variants was assessed by DSC with or without the liability of the N-glycosylation sequence. The first transition _Tm (attributed to the domain of the scFv) of F3'-1297 and F3'-1306 TriNKETs was significantly higher (66.5°C-67.5°C) than the other F3'TriNKETs. Furthermore, the T _onsets of these two molecules were significantly superior compared to other F3'TriNKETs tested. The only difference between these two F3'-TriNKETs is the orientation of the scFv. F3'-1297 (VH-VL direction) and F3'-1306 (VL-VH direction).

To determine whether the human residue at position (L43) is responsible for the increased thermal stability, both F3'-1306 and F3'-1297 replaced murine residue I (L43) with the original Reverted to human framework residue A (L43). In addition, mouse Ile at position L43 of F3'-1295 was replaced with human Ala and then made recombinantly. A new second generation humanized TriNKET derived from F3'-1295 was named F3'-1602 or F3'-1602 TriNKET.

Thermal stability of F3'-1295 and second generation F3'-1602 TriNKETs was assessed by DSC in PBS (Figure 25). Based on the T _m of the first transition, the scFv of F3′-1602 was thermostabilized at 7.5° C. compared to F3′-1295, shown in Table 20. This stabilization is due to Ile/Ala mutations. Therefore, F3'-1602 met the T _m1 cutoff above 65°C.

To understand whether the I(L43)A substitution affected the TriNKET affinity for hCLEC12A, the binding of hCLEC12A to F3'-1295 and F3'-1602 was measured by SPR (Biacore) at 37°C ( 26) and using the methods described in Example 1. Kinetic constants and equilibrium binding affinities are listed in Table 21. Both TriNKETs have very similar off-rates, but F3'-1602 exhibits a K _D approximately two-fold lower due to its faster on-rate. Thus, the I(L43) substitution affected the _KD .

F3′-1295 and F3′-1602 were compared to parental Ba/F3 cells for their ability to bind isogenic Ba/F3 cells expressing human CLEC12A (as shown in FIG. 27A and Table 22). data listed in ) were tested using the methods described in Examples 5 or 6. F3'-1602 was able to bind to hCLEC12A+Ba/F3 with approximately 2-fold lower EC50 values compared to F3'-1295, although the maximum binding MFI was similar. Binding to parental Ba/F3 cells lacking expression of CLEC12A was detected by either F3'-1295 or F3'-1602 (Fig. 27B), suggesting high specificity of TriNKET binding to hCLEC12A.

F3'-1295 and F3'-1602 were further tested for their ability to bind to HL60 (Figure 27C) and PL21 (Figure 27D) AML cancer cell lines using methods described in Examples 5 or 6. was done. As shown in Table 23, F3'-1602 was able to bind to both cell lines with lower EC50 values compared to F3'-1295 and the maximal binding MFIs were similar.

The potency of F3'-1295 and F3'-1602 TriNKETs was evaluated in a KHYG-1-CD16aV-mediated cytotoxicity assay. The results are shown in FIG. Both F3′-1602 and F3′-1295 induced potent lysis of PL21 (FIG. 28A) and HL60 (FIG. 28B) AML cancer cells, both with similar EC50 and % maximal lysis as shown in Table 23. indicate.

The hydrophobic properties of F3′-1295 and F3′-1602 TriNKETs were evaluated by analytical Hydrophobic Interaction Chromatography (HIC) and the results are shown in Table 24. Trastuzumab (Roche) was used as a representative control for therapeutic antibodies. The retention times (RTs) of both TriNKETs were similar to that of trastuzumab, suggesting a predicted low aggregation propensity for both TriNKETs.

The experimental isoelectric points (pI) of F3′-1295 (FIG. 29A) and F3′-1602 (FIG. 29B) were assessed by capillary isoelectric focusing (cIEF), as described in Example 15. rice field. Both constructs exhibited nearly identical pIs. The major peak of F3'-1295 has a pI of 8.73 and the major peak of F3'-1602 has a pI of 8.81 (Table 25).

When developing protein therapeutics, it is necessary to assess the drug's off-target effects. Flow cytometry-based multispecific reagent (PSR) assays allow the measurement of antibodies that are likely to bind non-specifically. F3'-1295 and F3'-1602 were tested for non-specific binding to detergent-solubilized CHO cell membrane protein preparations in a PSR assay using the method described in Example 12. Both humanized F3′-1602 and F3′-1295 did not bind PSR (no signal shift to the right) and showed very similar profiles to the PSR control trastuzumab, as shown in FIG. suggesting a high specificity of Rituximab was used as a positive control in this assay. F3'-1602 was selected for further comparison with additional TriNKETs described.

The effects of different TriNKET formats were experimentally tested for varying efficacy. To obtain CLEC12A TriNKET in F3 format, humanized 16B8. C8 was converted to chains VH-CH1-Fc and VL-CL using the same humanized variants used for F3'-1602, while NKG2D A49M-ImAb was converted to VH-VL-Fc. was done. This protein was called AB0010 or F3-1602.

The binding affinity of human CLEC12A-His to AB0010 (F3 TriNKET format) and F3'-1602 (F3'TriNKET format) was assessed by SPR (Biacore) at 37°C. Kinetic constants and equilibrium binding affinities are shown in Table 26 and raw data and fits are shown in FIG. The overall binding affinities of both constructs were within 2-fold difference, as evidenced by _KD , with AB0010 forming a slightly stronger complex with CLEC12A compared to F3'-1602.

Different formats of TriNKET, F3′-1602 (F3′) and AB0010 (F3), were tested for their ability to bind to Ba/F3 cells expressing human CLEC12A (FIG. 32A) and AML cancer cell lines (FIG. 32B). . The EC50 value of F3'-1602 was superior to AB0010 in HL-60 cells and decreased approximately two-fold (Table 27). Neither AB0010 nor F3'-1602 showed binding to parental Ba/F3 cells, indicating high specificity of binding to CLEC12A (Fig. 32C).

The potency of AB0010 and F3'-1602 TriNKET was evaluated in the KHYG-1-CD16aV-mediated cytotoxicity assay shown in Figure 33A. F3'-1602 induced lysis of HL60 and PL21 AML cancer cells with EC50 values 2-fold superior to AB0010 (Table 28). Both the F3 (AB0010) and F3'(F3'-1602) TriNKETs were shown to be superior to the corresponding humanized parental mAb (AB0037-001).

The potency of AB0010 and F3'-1602 TriNKET was evaluated in a human primary NK cell-mediated cytotoxicity assay (Fig. 33B). F3'-1602 (F3' format) induced lysis of PL21 AML cancer cells with an EC50 value 2-fold lower than AB0010 (F3 format) (Table 29).

In both the KHYG-1-mediated cytotoxicity assay and the primary NK cell-mediated cytotoxicity assay, F3'-1602 (F3' format) showed an approximately 2-fold superior EC50 to AB0010 (F3 format). whereas the overall maximal kill rate was observed to be unaffected. In accelerated stability and forced degradation studies, AB0010 was structurally less stable compared to F3'-1602 (F3'-1602) (see Example 16). Therefore, CLEC12A TriNKET in F3' format (F3'-1602) was selected for further study.

9F11. To generate the B7 TriNKET, 9F11. All 12 scFv variants of B7 were combined with the A49M-I NKG2D Fab arms to create the humanized F3'TriNKET. The affinity of 10 semi-purified (Protein A) TriNKETs for hCLEC12A was evaluated by SPR in the screening mode shown in Table 30. Three (AB0190, AB0193, and AB0196) 9F11. B7-based F3'-TriNKET shows heterogeneous binding and cannot fit a reliable 1:1 kinetic model. The binding kinetics of the remaining seven TriNKETs were compared with the chimeric parental mouse 9F11. Similar to B7 mAb, suggesting that neither humanization nor conversion of Fab to scFv affected affinity for hCLEC12A.

Nine fully purified humanized 9F11.hCLEC12A+Ba/F3 and HL60 AML cancer cell lines shown in FIGS. 34A and 34B. Binding of B7 TriNKET was tested by FACS. AB0190, AB0193, and AB0196 exhibited poor binding EC50 values on both cell lines (Table 31), which correlates with the poor SPR behavior described in Table 30. The EC50 values of the remaining 6 clones were similar, showing correlation with the SPR data.

9F11. The efficacy of B7-based TriNKET was evaluated in a KHYG-1-CD16aV cell-mediated cytotoxicity assay (Figure 35 and Table 32). AB0190, AB0193, and AB0196 displayed minimal activity. AB0189 and AB0192 showed the highest potency among other TriNKET candidates. AB0192 TriNKET is 9F11. Humanization efforts of B7 emerged as the lead TriNKET.

The potency of AB0192 was evaluated in the KHYG-1-CD16aV cell-mediated cytotoxicity assay compared to F3'-1602 (Figure 36A). F3'-1602 showed improved efficacy over AB0192 in both EC50 (0.4 nM and 1.3 nM, respectively) and maximal HL-60 cell lysis rate (41% and 22%, respectively).

The potency of AB0192 and F3'-1602 TriNKET was further evaluated in a human primary NK cell-mediated cytotoxicity assay (Fig. 36B). F3'-1602 and AB0192 induced lysis of HL60 AML with similar EC50 and maximum % cell killing (Table 33).

AB0192 meets TriNKET pre-developability criteria: high affinity for hCLEC12A, efficient binding to AML cells, high potency of AML cell killing in primary NK cell-mediated cytotoxicity assay, high thermostability, T _m1 >65° C., low hydrophobicity, 8.0<PI<9.0, expression level in transient transfection similar to monoclonal antibody, clean PSR (Table 33).

F3'-1602 showed better potency, more human-likeness and less potential sequence liability compared to AB0192. The structural stability of F3'-1602 was confirmed in an extensive panel of accelerated stability and forced degradation studies described in Example 15. Therefore, F3'-1602 was selected for further development.

実施例６．細胞発現ヒトがん抗原へのＴｒｉＮＫＥＴ結合の評価 F3'-1602 and its corresponding humanized mAb (h16B8.C8) were evaluated for cytotoxic cell killing via primary NK cells. F3′-1602 showed potent killing of PL21 (FIG. 37A) and THP-1 (FIG. 37B) AML cancer cell lines, whereas the corresponding monoclonal antibodies had significantly reduced potency (Table 34).

Example 6. Evaluation of TriNKET binding to cell-expressed human cancer antigens

Isogenic Ba/F3 cells expressing hCLEC12A and human cancer cell lines HL60 and PL21 expressing human CLEC12A were used to assess tumor antigen binding of TriNKET and mAbs targeting CLEC12A.

AB0089 is an F3'TriNKET derived from the same binding domain as the parent mAb AB0305. AB0192 is an F3'TriNKET derived from the same binding domain as the parent mAb AB0192. AB0237 is an F3'TriNKET, also referred to herein as hF3'-1602 or F3'-1602 TriNKET, derived from scFv-1602. AB0300 (also called F3'-1602si or AB0237si) is also an F3'TriNKET with the same sequence as AB0237, but with mutations in the CH2 domain that eliminate Fc-gamma receptor binding. F3'TriNKET generally showed weaker binding compared to the parental mAb.

To assess whether the antigen-binding site that binds NKG2D and the effector function of the Fc contribute to the cytotoxicity of AB0237, two TriNKET variants were constructed. The first variant contains mutations in the light chain variable domain of the A49 antigen binding site that binds NKG2D. As a result, this variant does not bind human NKG2D. The amino acid sequence of the mutated light chain variable domain is DIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLIYEASSTKSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGGGTKVEIK (SEQ ID NO: 282). The amino acid sequence of this first variant is otherwise identical to that of AB0237. A second variant contains mutations in the Fc domain. Specifically, each polypeptide chain of the Fc domain contains the L234A/L235A/P329G substitutions (numbered under the EU numbering system) reported to reduce binding of Fc to Fcγ receptors. . The amino acid sequence of this second variant is otherwise identical to that of AB0237. F3'-palivizumab contains the same NKG2D and CD16 binder sequences as F3'-1602 and an scFv that binds to an unrelated target not expressed in either of the two cancer cell lines tested. EC50 values are shown in Table 35.

The cells were then incubated with a fluorophore-conjugated anti-human IgG secondary antibody and analyzed by flow cytometry. Mean fluorescence intensity (MFI) values were normalized to secondary antibody-only controls to obtain fold over background (FOB) values.

AB0237 shows high affinity binding to two AML cell lines tested, namely HL60 (Figure 38A) and PL21 (Figure 38B). The EC50 for HL60 was 2.2 nM and the EC50 for PL21 was 1.2 nM.

For two of the three binders, F3'TriNKET showed higher target cell loading compared to its parental mAb. AB0237, AB0300, and AB0237 NKG2D dead arm mutational variants were compared with their parental TriNKET AB0237 for Ba/F3-hCLEC12A (Figure 39A), HL-60 (Figure 39B), and PL-21 (Figure 39C). showed similar binding.
Example 7. Evaluation of TriNKET surface retention for cell-expressed human cancer antigens

Human cancer cell lines HL60 and PL21 expressing human CLEC12A were used to assess surface retention of CLEC12A-TriNKET after incubation with cells. PL21 or HL60 cells in replicate plates were incubated with TriNKET or mAbs as indicated. The first plate was moved to 37°C for 2 hours and the second plate was incubated on ice for 20 minutes. After each incubation period, cells were washed. Surface-bound TriNKET or mAbs were detected using fluorescent secondary antibodies for flow cytometry.
Surface retention was calculated as follows.
% surface retention = (sample MFI 2 hours/sample MFI 20 minutes)) ^* 100%

Figures 40A and 40B show cell surface retention of TriNKET bound to CLEC12A+ cell lines, PL21 and HL60, respectively. The monovalently conjugated TriNKETs, AB0089, AB0147, AB0192, and AB0237, showed high surface retention at 2 hours on both PL21 and HL60 cell lines. At both PL21 and HL60, the bivalent binding mAb AB0037 showed lower surface retention at 2 hours compared to TriNKET.
Example 8. Human NK cell cytotoxicity assay

Target cell lysis was measured by the DELFIA cytotoxicity assay. Briefly, human cancer cell lines expressing CLEC12A were harvested from culture, washed with HBS, and labeled with BATDA reagent (Perkin Elmer AD0116) at 10 ⁶ /mL in growth medium. was resuspended in The manufacturer's instructions were followed for target cell labeling. After labeling, cells were washed three times with HBS and resuspended in medium at 0.5-1.0×10 ⁵ /mL. 100 μl of BATDA-labeled cells were added to each well of a 96-well plate. Monoclonal antibodies against CLEC12A or TriNKET were diluted in culture medium and 50 μl of diluted mAb or TriNKET was added to each well.

To prepare NK cells, PBMCs were isolated from human peripheral blood buffy coat using density gradient centrifugation, washed and prepared for NK cell isolation. NK cells were isolated using a negative selection technique with magnetic beads. The purity of isolated NK cells was usually greater than 90% CD3-CD56+. Isolated NK cells were rested overnight and harvested from culture. Cells were then washed and resuspended in culture medium at a concentration of 10 ⁵ -2.0×10 ⁶ /mL with an effector to target (E:T) ratio of 5:1. 50 μl of NK cells were added to each well of the plate for a total culture volume of 200 μl. The plates were incubated for 2-3 hours at 37°C with 5% _CO2 .

After incubation, plates were removed from the incubator and cells were pelleted by centrifugation at 200 xg for 5 minutes. 20 μl of culture supernatant was transferred to a clean microplate and 200 μl of room temperature europium solution (Perkin Elmer C135-100) was added to each well. The plates were protected from light and incubated on a plate shaker at 250 rpm for 15 minutes before reading using the SpectraMaxi3X instrument.

Spontaneous release of substances capable of forming fluorescent chelates with europium was measured in target cells incubated in the absence of NK cells. Maximal release of such substances was determined with target cells lysed with 1% Triton®-X. % specific lysis was calculated as follows:
% specific lysis = ((experimental release - spontaneous release) /
(maximal release - spontaneous release)) ^* 100%.

Figures 41A and 41B show CLEC12A-TriNKET-mediated killing of PL21 and HL60 target cells by resting human NK cells, respectively. Monoclonal antibodies targeting AB0037 CLEC12A showed little enhancement of NK cell-mediated lysis of PL21 and HL60 target cells. All three CLEC12A-targeting TriNKETs (AB0237, AB0192, and AB0089) showed superior lysis of target cells compared to CLEC12A-targeting monoclonal antibodies. EC50 values are shown in Table 36.

実施例９．初代ヒトＣＤ８＋Ｔ細胞細胞傷害性アッセイ To confirm the cytotoxicity findings, a second NK cell line, KHYG-1-CD16aV, was engineered to stably express CD16aV and NKG2D and assayed as described above. Cytotoxicity of PL21 is shown in Figure 42A; cytotoxicity of HL60 is shown in Figure 42B. EC50 and maximal lysis values were derived from the cell lysis curves by GraphPad Prism software using a four parameter logistic nonlinear regression curve fitting model (Table 37). F3′-1602 exhibits a sub-nanomolar EC50 and highly efficient maximal lysis in the KHYG-1CD16A-mediated cytotoxicity assay.

Example 9. Primary human CD8+ T cell cytotoxicity assay

Target cell lysis was measured by the DELFIA cytotoxicity assay as described in Example 8, except that CD8+ T cells were used as immune effector cells. CD8+ T cells were prepared as follows. Human PBMC were isolated from human peripheral blood buffy coat using density gradient centrifugation using Lymphoprep and SepMate 50 according to the manufacturer's instructions. Isolated PBMCs were stimulated with 1 μg/mL ConA (IL-2 culture supplement) in culture medium at 37° C. for 18 hours. ConA was then removed and PBMCs were cultured with 25 units/mL IL-2 at 37° C. for 4 days. CD8+ T cells were purified using magnetic bead-based negative selection technology (EasySep™ Human CD8+ T cell isolation kit) according to the manufacturer's instructions. CD8+ T cells were cultured in medium containing 10 ng/mL IL-15 at 37° C. for 6-13 days prior to use in cytolytic assays.

As shown in Figure 43, AB0237 TriNKET significantly enhanced the ability of CD8 T cells to lyse target cells, but not the parental AB0037 mAb. AB0237 TriNKET dose-dependently increased CD8+ T cell-mediated cytolysis.

TriNKET variants were constructed to assess whether the antigen-binding site that binds NKG2D and the effector function of Fc contribute to the cytotoxicity of AB0237 TriNKET. This variant contains mutations in the light chain variable domain of the A49 antigen binding site that binds NKG2D. As a result, this variant does not bind to human NKG2D. The amino acid sequence of the mutated light chain variable domain is DIQMTQSPSTLSASVGDRVTITCRASNSISSWLAWYQQKPGKAPKLLIYEASSTKSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYDDLPTFGGGTKVEIK (SEQ ID NO: 282). The amino acid sequence of this first variant is otherwise identical to that of AB0237 TriNKET.

The ability of variants to induce NK cell-mediated lysis of target cells was assessed using the DELFIA cytotoxicity assay described above. As shown in Figure 43, mutation of the NKG2D targeting domain significantly reduced cytotoxicity. This result suggested that both binding to NKG2D and binding to Fcγ receptors contributed to the cytotoxic activity of AB0237 TriNKET.

Example 10. Evaluation of TriNKET or mAb Binding to Human Whole Blood The ability of AB0237 and mAb AB0037 to bind to different types of blood cells was evaluated. Briefly, human whole blood was incubated with AB0237 (grey), mAb AB0037 (white), or human IgG1 isotype control antibody (black). Blood cells were analyzed by flow cytometry and binding of AB0237, mAb AB0037, or an isotype control antibody was detected using a fluorophore-conjugated anti-human IgG secondary antibody.

As shown in Figure 44, staining was observed for the percentage of monocytes, granulocytes and dendritic cells in whole blood for AB0237 and its parent mAb AB0037.
Example 11. Binding of F3′-1602 TriNKET to Fc Receptors: Characterization of Label-Free Surface Plasmon Resonance

The binding affinities of F3'-1602 TriNKETs to recombinant human and cynomolgus monkey Fcγ receptors were determined and compared to that of the IgG1 isotype control trastuzumab. The binding affinities of F3'-1602 TriNKET to human and cynomolgus monkey (cyno) FcRn at pH 6.0 and pH 7.4 were also determined.

FcγR binding Human and cynomolgus monkey CD64, high and low affinity alleles of CD16a (V158 and F158, respectively), two alleles of CD32a (H131 and R131), CD16b, CD32b and cynomolgus CD16 were Captured via biotin on a primed Biacore chip prepared according to instructions. Prior to FcγR binding experiments requiring high starting concentrations of F3′-1602 TriNKET and trastuzumab, samples were buffered in 1×HBS-EP+SPR running buffer by 3 serial dilution and concentration cycles using 0.5 mL concentration units. Liquid exchanges were performed to dilute the original buffer components over 300-fold while maintaining high protein concentration. Protein concentration was measured at absorbance at A280 on a NanoDrop instrument using the appropriate theoretical molar extinction coefficient of the sample (198405 M ⁻¹ cm ⁻¹ for F3′-1602 TriNKET and standard IgG settings for trastuzumab). bottom.

Each FcγR binding experiment was performed in multiple cycles utilizing 2-fold serial dilutions of F3′-1602 TriNKET or trastuzumab. A 0 nM blank cycle at the start of each concentration series and a chip surface without receptor was used as a reference for no signal. Each experimental cycle included association, dissociation, and regeneration steps. All steps were performed at a flow rate of 30 μL/min and at 25°C. Analyte (F3′-1602 TriNKET or trastuzumab) concentrations, association and dissociation times, and regeneration parameters were FcγR-specific (Table 38).

The geometry of raw sensorgrams representing F3'-1602 TriNKET and trastuzumab-bound recombinant human CD64 allowed the data to be fitted with a 1:1 kinetic fitting model. The black trace representing the 1:1 kinetic fit closely follows the actual sensorgram shown in color in FIG.

The χ ² error values calculated by the Biacore Insight evaluation software were less than 1.9% of the calculated R _max , representing an excellent fit. Table 39 summarizes the locomotion rate and human CD64 affinity values of F3'-1602 TriNKET and trastuzumab.

Binding of F3'-1602 TriNKET to CD32aH131 was determined as described above and is shown in FIG. This data was fitted to a steady-state affinity fitting model. Affinity values are summarized in Table 40.

Binding of F3'-1602 TriNKET to the human CD32A R131 allele (FCγRIIaR131) was determined as described above and is shown in FIG. The data were fitted to a steady-state affinity fitting model; affinity values are summarized in Table 41.

Binding of F3'-1602 TriNKET to human CD16A high affinity allele V158 (FCγRIIIa V158) was determined as described above and is shown in FIG. The data were fitted to a 1:1 kinetic fitting model; kinetic parameters and affinity values are summarized in Table 42. The affinity of F3'-1602 TriNKET for both alleles is comparable to that of trastuzumab for the same receptor (less than 3-fold difference).

Binding of F3'-1602 TriNKET to the human CD16A low affinity allele F158 (FCγRIIIa F158) was determined as described above and is shown in FIG. Data were fitted to a steady-state affinity fitting model; affinity values are summarized in Table 43.

Binding of F3'-1602 TriNKET to human CD32b (FCγRIIB) was measured as described above and is shown in FIG. Data were fit to a steady-state affinity fitting model; affinity values are summarized in Table 44.

Binding of F3'-1602 TriNKET to human CD16b (FCγRIIIB) was measured as described above and is shown in FIG. Data were fitted to a steady-state affinity fitting model. Affinity values are summarized in Table 45.

Binding of F3′-1602 TriNKET to cynomolgus monkey (cyno) CD16 was measured as described above and is shown in FIG. Data were fitted to a steady-state affinity fitting model; affinity values are summarized in Table 46.

Binding of F3'-1602 TriNKET to human FcRn was measured at pH 6.0 as described above and is shown in FIG. This data was fitted to a steady-state affinity fitting model. Affinity values are summarized in Table 47.

Binding of F3′-1602 TriNKET to cynomolgus monkey (cyno) FcRn was measured at pH 6.0 as described above and is shown in FIG. This data was fitted to a steady-state affinity fitting model. Affinity values are summarized in Table 48.

The binding of F3'-1602 TriNKET to human and cynomolgus monkey (cyno) FcRn was measured at pH 7.4 as described above. No quantifiable binding was observed.

F3′-1602 TriNKET and the IgG1 isotype control trastuzumab showed no physiologically meaningful difference (less than a 3-fold difference), indicating that the human and cynomolgus CD64 (FCγRI), CD16 (FCγRIII) groups tested Their binding to the recombinant receptor is comparable (Table 49). F3′-1602 TriNKET and trastuzumab are similar in their binding to the human CD32 (FCγRII) receptor (less than 1.2-fold difference) (Table 49). F3'-1602 TriNKET is similar to trastuzumab in its affinity for human and cynomolgus monkey FcRn at pH 6.0. F3'-1602 TriNKET and trastuzumab showed no detectable binding at the concentrations tested at pH 7.4 (Table 50).

FcRn Binding Recombinant human or cynomolgus monkey (cyno) FcRn was directly immobilized via amine coupling at densities of 169-216 RU. F3′-1602 TriNKET and commercial trastuzumab as an IgG1 assay control were buffer exchanged to MBS-EP+, pH 6.0 as above prior to SPR binding experiments. Each FcRn binding experiment consisted of multiple cycles utilizing varying concentrations of the analyte. Two-fold dilution series (12 μM-0.023 μM) of F3′-1602TriNKET and trastuzumab were used. As a no signal, negative control, a blank amine-coupled modified chip surface lacking FcRn was used, with a blank cycle of 0 nM placed at the beginning of each concentration series. Each experimental cycle included association, dissociation, and regeneration steps. This association step consisted of a 60 second analyte injection followed by a 60 second dissociation step. Surface regeneration (60 sec injection of HBS-EP+buffer, pH 7.4) completed each kinetic cycle. All steps were performed at a flow rate of 30 μL/min and 25°C. MBS-EP+ buffer, pH 6.0 was used as running buffer. This data was fitted to a steady-state affinity model using the Biacore Insight evaluation software. The ^chi2 (calculated by the software) associated with the calculated _Rmax was used to assess the goodness of fit.

Example 12. Molecular Format and Design, Structure, Affinity, Potency, Specificity, Cross-Reactivity Analysis of F3′-1602 TriNKET Surface Plasmon Resonance (SPR)
The binding affinities of F3′-1602 TriNKET to recombinant human CLEC12A (hCLEC12A) or cynomolgus monkey (cyno) CLEC12A (cCLEC12A) were measured by SPR using a Biacore 8K instrument at physiological temperature of 37°C. Briefly, human Fc-specific antibodies were covalently immobilized onto the carboxymethyldextran matrix of CM5 biosensor chips at a density of approximately 8000-10000 resonance units (RU) to generate anti-hFc IgG chips. . The F3′-1602 TriNKET sample was injected over the anti-hFc IgG chip at a flow rate of 10 μL/min for 60 seconds to achieve a capture level of approximately 250 RU. hCLEC12A-His or cCLEC12A-His were serially diluted (100 nM to 0.14 nM) in 3-fold dilutions in running buffer and injected over the captured test substances at a flow rate of 30 μl/min. Association was monitored for 300 seconds and dissociation for 900 seconds. The surface was regenerated between cycles by injecting three pulses of 10 mM glycine-HCl (pH 1.7) at 100 μL/min for 20 seconds.

Kinetic constants and equilibrium binding affinities are shown in Table 51 and raw data and fits are shown in FIG. This molecule binds strongly to CLEC12A and weakly to NKG2D and CD16a. The complex between F3′-1602 TriNKET and human CLEC12A is potent as evidenced by a slow dissociation constant of 4.94±0.09×10 ⁻⁴ s ⁻¹ . The equilibrium binding affinity K _D of F3'-1602 TriNKET was 0.59±0.01 nM.

In addition, the polymorphic variant CLEC12A-K244Q is prevalent in approximately 30% of the population. The binding of F3'-1602 TriNKET to CLEC12A-K244Q was examined by SPR to compare its affinity with wild-type CLEC12A. As shown in Table 52, the kinetics of binding of F3'-1602 TriNKET to wild-type and K244Q variants of CLEC12A are similar.

The binding affinity of F3'-1602 TriNKET to recombinant human NKG2D was measured by SPR using a Biacore 8K instrument at physiological temperature of 37°C. Briefly, mouse (mFc)-specific antibodies were covalently immobilized onto the carboxymethyldextran matrix of CM5 biosensor chips at densities of approximately 8000-10000 RU to generate anti-mFc IgG chips. mFc-tagged human NKG2D was captured onto an anti-mFc IgG chip at a flow rate of 10 μL/min for 60 seconds to achieve a capture level of approximately 35 RU. F3′-1602 TriNKET was buffer exchanged into running buffer and serially diluted (5000 nM to 9.78 nM) in 2-fold dilutions with 1×HBS-EP+. F3'-1602 TriNKET was injected at a flow rate of 30 μl/min over the captured test material. Association was monitored for 60 seconds and dissociation was monitored for 60 seconds. The surface was regenerated between cycles by injecting three pulses of 10 mM glycine-HCl (pH 1.7) at 100 μL/min for 20 seconds.

Figure 56 shows F3'-1602 TriNKET binding sensorgrams to human NKG2D. To obtain equilibrium affinity data, two different fits were utilized: a steady-state affinity fit and a kinetic fit. Kinetic constants and equilibrium affinity constants are shown in Table 54. The dissociation rate constant was (1.30±0.03)×10 ⁻¹ s ⁻¹ . The equilibrium affinity constants (K _D ) obtained by kinetic and steady-state affinity fits were 721±21 nM and 727±22 nM, respectively.

Cross-reactivity of F3′-1602 TriNKET with cynomolgus monkey (cyno) NKG2D was further investigated by SPR as described in Examples 5 or 6. F3′-1602 TriNKET was tested at 37° C. for binding to recombinant cynomolgus monkey (cyno) NKG2D (cNKG2D) extracellular domain (ECD) by SPR (Biacore) (FIG. 57). Recombinant mFc-tagged cynomolgus monkey (cyno) NKG2D dimers were used for this experiment, as NKG2D is dimeric in nature. Binding affinities are shown in Table 55. Binding affinities were obtained from a kinetic fit (K _D 839±67 nM) and a constant fit (K _D 828±88 nM) and were similar to those observed for hCLEC12A (kinetic fit _KD 721 ± 21 nM obtained at ).

Simultaneous engagement of CLEC12A and NKG2D F3′-1602 TriNKET was diluted in 1×HBS-EP+ buffer containing 0.1 mg/mL BSA and captured onto the anti-human Fc surface of a CM5 chip at a flow rate of 5 μL/min for 60 seconds to A capture level of 150-250 RU was achieved. The net difference between the baseline signal and the signal after completion of F3'-1602 TriNKET injection representing the amount of F3'-1602 captured was recorded. hCLEC12A-His (100 nM) or mFc-hNKG2D (7 μM) was injected at 30 μL/min over the captured F3′-1602 TriNKET for 90 seconds until saturation was reached. Immediately following this injection, a preincubated mixture of hCLEC12A-His (100 nM) and mFc-hNKG2D (7 μM) was injected at a flow rate of 30 μL/min for 90 seconds using the ABA injection command of the Biacore 8K control software. (the second target was pre-mixed with the first target to ensure that all binding sites of the first target were occupied). The chip was regenerated by two 20 second pulses of 10 mM glycine (pH 1.7) at 100 μL/min. This experiment was performed at 37° C. to mimic physiological temperature. The average relative binding stoichiometry of each target bound to the captured F3′-1602 TriNKET already saturated with the other target (injected second) was higher than that of the unoccupied F3′-1602 TriNKET was expressed as a percentage of the total capacity bound to

Target binding sensorgrams, such as shown in Figure 58, show that CLEC12A domain occupancy does not interfere with NKG2D binding and that NKG2D domain occupancy does not interfere with CLEC12A binding. The similarity in shape of each sensorgram segment showing binding of each target to free F3′-1602 TriNKET and F3′-1602 TriNKET saturated with the other target indicates that the kinetic parameters of F3′-1602 It is suggested that it is not significantly affected by the target occupancy state of the TriNKET molecule. For example, the shape of the CLEC12A binding segment in the sensorgrams is similar in both panels. A saturating concentration of NKG2D was maintained throughout the experiment, which is shown in the bottom panel due to the fast dissociation rate of the target. Furthermore, the lack of effect on the relative stoichiometry of each target binding (when compared to binding to unoccupied F3'-1602) suggests that the NKG2D and CLEC12A binding sites on F3'-1602 TriNKET are means completely independent (Table 56).

Synergy of NKG2D-CD16a Synergistic NKG2D and CD16a binding was assessed by SPR. 233 RU of hNKG2D alone, 618 RU of CD16a F158 allele alone, and 797 RU of a mixture of hNKG2D and CD16a (F158) were amino-coupled to the surface of a CM5 series S Biacore chip. 2.8 μMF 3′-1602 TriNKET or trastuzumab were injected at 10 μL/min for 150 seconds. The dissociation phase was observed for 180 seconds when no regeneration was required, or 1500 seconds when spontaneous regeneration of the surface (almost complete dissociation of the analyte) was required between cycles at the same flow rate.

As shown in Figure 59, F3'-1602 TriNKET binds only CD16a or NKG2D with low affinity, whereas F3'-1602 TriNKET injected into the mixed surface simultaneously binds CD16a and NKG2D with a much slower off-rate. It appears as the cohesive strength of the bond (Fig. 59A). The experimental control trastuzumab does not bind to immobilized NKG2D and shows the same apparent off-rate when injected over both mixtures (CD16a+NKG2D) and CD16a-only surfaces (FIG. 59B). This data indicates that F3'-1602 TriNKET strongly engages both CD16a and NKG2D.

Binding of Unrelated Recombinant Proteins and Cell Binding Specificity Specificity for CLEC12A was tested by SPR against 5 different unrelated proteins at concentrations as high as 500 nM and is shown in FIG. The positive control (CLEC12A) at a concentration of 100 nM showed binding of 50 RU (Fig. 60A), whereas non-specific binding was observed to any of the irrelevant recombinant targets (Fig. 60B, Fig. 60C). it wasn't. Specificity was also tested against proteins expressed on the surface of the Ba/F3 cell line. Although no non-specific binding of F3'-1602 to the Ba/F3 parental cell line was observed at a concentration of 333 nM (Fig. 60E), Ba/F3 engineered to express CLEC12A was used as a positive control. Cell lines showed a significant shift by flow cytometry (Fig. 60D).

Non-Specific Binding to Multispecific Reagents (PSR) A flow cytometry-based PSR assay allows the exclusion of antibodies that likely bind non-specifically to irrelevant proteins. As part of the developmental evaluation, F3'-1602 was tested for non-specific binding to preparations of detergent-solubilized membrane protein in the PSR assay (Figure 61). The PSR assay correlates well with cross-interaction chromatography, a surrogate for antibody solubility, as well as a surrogate for baculovirus particles, enzyme-linked immunosorbent assays, in vivo clearance (Xu et al., (2013) ．Ａｄｄｒｅｓｓｉｎｇ ｐｏｌｙｓｐｅｃｉｆｉｃｉｔｙ ｏｆ ａｎｔｉｂｏｄｉｅｓ ｓｅｌｅｃｔｅｄ ｆｒｏｍ ａｎ ｉｎ ｖｉｔｒｏ ｙｅａｓｔ ｐｒｅｓｅｎｔａｔｉｏｎ ｓｙｓｔｅｍ：ａ ＦＡＣＳ－ｂａｓｅｄ，ｈｉｇｈ－ｔｈｒｏｕｇｈｐｕｔ ｓｅｌｅｃｔｉｏｎ ａｎｄ ａｎａｌｙｔｉｃａｌ ｔｏｏｌ．Ｐｒｏｔｅｉｎ ｅｎｇｉｎｅｅｒｉｎｇ ｄｅｓｉｇｎ ａｎｄ ｓｅｌｅｃｔｉｏｎ，２６，６６３－６７０）。

50 μL of 100 nM TriNKET or control mAb in PBSF were incubated with 5 μL of pre-washed Protein A Dynabeads slurry (Invitrogen, Cat#10001D) for 30 min at room temperature. Magnetic beads bound to TriNKET or mAb were left on the magnetic rack for 60 seconds and the supernatant was discarded. Bound beads were washed with 100 μL PBSF. Beads were incubated with 50 μL of biotinylated PSR reagent diluted 25-fold from stock for 20 min on ice (Xu et. al., (2013) Protein engineering design and selection, 26, 663-670). Samples were placed on a magnetic rack, supernatant discarded and washed with 100 μL PBSF. Secondary FACS reagents for detecting binding of biotinylated PSR reagents to TriNKET or control mAbs were made up as follows: 1:250 μL streptavidin-PE (Biologend, Catalog No. 405204) and 1:100. donkey anti-human Fc was combined with PBSF. 100 μL of secondary reagent was added to each sample and allowed to incubate on ice for 20 minutes. The beads were washed twice with 100 μL PBSF and samples were analyzed on a FACS Celesta (BD). Two PSR controls, rituximab (PSR positive) and trastuzumab (PSR clean) were used in the assay.
High-Spec® in HuProt™ Human Proteome Assay

Cross-Reactivity Assay Protein array technology was used to examine the specificity of F3'-1602 TriNKET. The HuProt™ Human Proteome Microarray provides the largest database of individually purified full-length human proteins on a single microscope slide. An array consisting of 22,000 full-length human proteins was generated from yeast S. cerevisiae. cerevisiae, and then printed in duplicate on microarray slides, allowing high-throughput profiling of thousands of interactions.

The specificity of F3′-1602 TriNKET was determined at concentrations of 0.1 μg/mL and 1 μg/mL against native Huprot human proteome arrays embedded in microslides from CDI laboratories (Baltimore, Md.) according to standard procedures. tested in

Figure 62 shows the relative binding (Z-score) of F3'-1602 TriNKET to human CLEC12A at 1 μg/mL compared to the entire human proteome microarray. A Z-score is the average binding score of two replicates of a given protein. For comparison purposes, the top 24 proteins with remaining background binding to F3'-1602 TriNKET are also provided in FIG. Table 57 shows the Z-score and S-score of F3'-1602 TriNKET for human CLEC12A and the top 6 proteins on the microarray. The S-score is the difference between the Z-score of a given protein and one rank next to it. Antibodies with an S-score greater than 3 for the top hits are considered highly specific for their target. Based on Z and S score criteria, F3'-1602 TriNKET showed high specificity to humans and lack of off-target binding in the HuProt™ Human Proteome Assay.

Molecular Modeling The anti-CLEC12A and anti-NKG2D binding arms of F3′-1602 TriNKET were compared to 377 post-Phase I biotherapeutic molecules using the Therapeutic Antibody Profiler (TAP) available at the SAbPred website. TAP used ABodyBuilder to generate a model of F3'-1602 TriNKET with side chains by PEARS. CDRH3 was constructed by MODELER due to its diversity.

Five different parameters were evaluated.
Full length of CDR Patch of surface hydrophobicity (PSH) near CDR Patch of positive charge (PPC) near CDR Patch of negative charge (PNC) near CDR Structural Fv charge symmetry parameter (sFvCSP)

These parameters of F3'-1602 TriNKET were then compared to the profile distribution of therapeutic antibodies to predict development feasibility and potential problems that could pose downstream challenges.

Figure 63 shows ribbon diagram models of CLEC12A binding scFv in three different orientations (upper panel) and the corresponding surface charge distributions in the same orientation (lower panel). The charge distribution of the anti-CLEC12A scFv is polarized ('top view', bottom panel), with negatively charged residues predominantly within CDRH3 and CDRL2. The heterogeneous distribution of electrostatic patches on the paratope is likely target-related and may reflect the complementarity of the charge distribution on the cognate epitope, which is similar to that of CLEC12A and F3. '-1602 TriNKET may contribute to the high affinity interaction between the scFv.

Figure 64 shows CDR length and surface hydrophobicity analysis of scFv CLEC12A targeting the arms of F3'-1602 TriNKET. The CDR length of the CLEC12A binding arm of F3'-1602 TriNKET is typical for late stage therapeutic antibodies.

Hydrophobicity of monoclonal antibodies is an important biophysical property related to their potential to be developed into therapeutic agents. Hydrophobic patch analysis of the antibody's CDRs predicts its behavior. The CLEC12A arm of F3'-1602 TriNKET is much less hydrophobic compared to other reference molecules. Based on modeling, there is no hydrophobic patch of significant size on the surface of the CLEC12A binding arm of F3'-1602 TriNKET.

The charge distribution of the anti-CLEC12A scFv is polarized, with negatively charged residues predominantly within CDRH3 and CDRL2 (as shown in Figure 64). Positively charged patches and charge symmetries are within the norm, but without wishing to be bound by theory, distinct negatively charged patches on the paratope are associated with targets and are associated with CLEC12A and CLEC12A. is hypothesized to reflect the complementarity of the charge distribution on the cognate epitope, which may contribute to the high-affinity interactions of .

The NKG2D-binding Fab arms of F3'-1602 TriNKET are shown in three different orientation ribbon views (Fig. 65) and their corresponding surface charge distributions in the same orientation are shown in Fig. 65 (bottom panel). . In contrast to the CLEC12A binding arm, the surface charge distribution of the NKG2D A49M-I arm is more evenly distributed.

Figure 66 shows patch CDR length and surface hydrophobicity analysis of the NKG2D targeting arm of F3'-1602 TriNKET. The analysis was performed using TAP and evaluated 377 late stage therapeutic antibodies. CDR length, hydrophobicity, positive/negative charge distribution, and Fc charge symmetry of the NKG2D-targeting arm of F3'-1602 TriNKET appear to be within the criteria of existing therapeutic antibodies. This modeling suggests that the F3'-1602 TriNKET does not pose any developability issues during the manufacturing process.

Hydrophobic Interaction Chromatography Hydrophobicity prediction data were confirmed by investigating the behavior of F3′-1602 TriNKET using analytical hydrophobic interaction chromatography (HIC), which showed that exposed hydrophobic Critical patching is a technology that relies on proteins that are prone to aggregation. Briefly, to perform HIC, TriNKET (5 μg protein) was injected in high salt buffer (100 mM sodium phosphate, 1.8 M ammonium sulfate, pH 6.5) to sample in a 5:4 ratio. prepared in Samples were analyzed using an Agilent 1260 Infinity II HPLC equipped with a Sepax Proteomix HIC Butyl-NP5 5uM column held at 25°C. The gradient was run from 0% low salt buffer (100 mM sodium phosphate, pH 6.5) to 100% low salt buffer over 6.5 minutes at a flow rate of 1.0 ML/min. Chromatograms were monitored at 280 nm. The retention time of F3′-1602 TriNKET on the analytical HIC column is shown in Table 58 and the HIC profile in FIG. Commercially available trastuzumab (Roche) was used as an example of a well behaved biologic and served as an internal control for the assay. F3'-1602 TriNKET has a retention time of 9.8 minutes compared to a retention time of 9.6 minutes for trastuzumab. Therefore, experimental hydrophobicity analysis suggests that the hydrophobicity of F3'-1602 TriNKET is amenable to further development.

Capillary isoelectric focusing (cIEF)
The experimental pI of F3'-1602 was obtained by cIEF as described in Example 15 (Figure 68). Commercial grade trastuzumab was included in the assay as an internal control. The cIEF profile of F3'-1602 TriNKET was typical of a monoclonal antibody, with a major peak at pI 8.80 (Table 59). The presence of minor amounts of acidic and basic species was also observed.

Immunogenicity Evaluation Immunogenicity evaluation was performed using EpiVax's EpiMatrix algorithm as described in Cohen et al. (2010) A method for individualizing the prediction of immunogenicity of protein vaccines and biological therapeutics: individualized T cell epitope measurements (iTEM) J.; Biomed. Biotechnol. 961752. For the sequence of the three chains of scFv-1602 F3', the T _reg- regulated Epimatrix protein scores ranged from -80 (not immunogenic) to 80 (highly immunogenic) for VH-CH1-Fc:-33. 39, VH-VL-Fc: -26.4 and VL-CL: -27.4. Therefore, the predicted risk of immunogenicity of scFv-1602 F3' appears to be low.

Example 13. F3′-1602 TriNKET Analysis of Putative Sequence Liability The amino acid sequences of the three polypeptide chains of F3′-1602 TriNKET were analyzed for putative sequence liability as described in Example 3. Estimated sequence liabilities are shown in Table 60.

Accelerated stability (4 weeks at 40° C.) and forced degradation studies were performed to examine the liability. The CDRH3 DP of VH-CH1-Fc was not altered in accelerated stability studies or forced degradation studies, so no further analysis was necessary. However, as described in Example 14, in accelerated stability studies, it was observed that alteration of DS in CDRH3 of scFv resulted in decreased CLEC12A binding and decreased potency of F3'-1602 TriNKET.

（配列番号２８３）

（配列番号２８４）及び

（配列番号２８５）は、親ｓｃＦｖ

（配列番号１３９）と比較して結合シグナルが弱いにもかかわらず、ｈＣＬＥＣ１２Ａへの結合を示したことが特定され、一方、バリアント

（配列番号２９０）、

（配列番号２９１）、及び

（配列番号２９２）が非バインダーとして特定された。結合分析に基づいて、バリアント

（（配列番号２８３）、ＡＢ００５３）及び

（（配列番号２８４）、ＡＢ００８５）のみを、哺乳動物の生成及びさらなる特性評価のために検討した。ＡＢ００５３及びＡＢ００８５のｈＣＬＥＣ１２Ａ－Ｈｉｓへの結合は、３７℃で表面プラズモン共鳴（ＳＰＲ）を使用して特徴付けた。ＤＳ配列のライアビリティを取り除くために導入された両方の変異は、ｈＣＬＥＣ１２Ａ－Ｈｉｓへの結合に影響を及ぼした（表６１）。図６９は、ＣＤＲＨ３の配列ライアビリティを取り除くために生成された酵母ライブラリーを使用したｈＣＬＥＣ１２Ａ－ｈｉｓへの結合のＦＡＣＳ分析を示している。このデータによって、ＤＳからＤＩ操作されたバリアント（ＡＢ００８５）の結合が完全に失われたことが示された。ＤＳ（ＡＢ００５３）の代わりにＤＡを導入しても、結合は完全には排除されなかったが、結合センサーグラムに明らかな不均一性が生じた。ＳＰＲ分析に続いて、ライアビリティを修復したバリアント（ＡＢ００５３及びＡＢ００８５）の効力を、ＫＨＹＧ－１－ＣＤ１６ａＶを介した細胞毒性アッセイで試験した（図７０、表６２）。ＡＢ００８５は、最大殺滅率の大幅な低下によって決定されるように、ＡＭＬ細胞を殺す能力を失った。ＡＢ００５３構築物のＥＣ５０がＦ３’－１６０２ ＴｒｉＮＫＥＴ（ＡＢ０２３７）対照と比較して約３倍に増大したが、最大殺滅の割合は影響を受けなかった。この知見は、ＳＰＲ実験で確立されたｈＣＬＥＣ１２Ａに対する全体的な親和性の３倍の変化と一致している（表６１）。 To replace the DS of CDRH3, yeast display was performed to identify alternative sequence motifs without the DS site. Three variants namely

(SEQ ID NO: 283)

(SEQ ID NO: 284) and

(SEQ ID NO:285) is the parent scFv

(SEQ ID NO: 139) was identified as showing binding to hCLEC12A, albeit with a weaker binding signal compared to (SEQ ID NO: 139), whereas the variant

(SEQ ID NO: 290),

(SEQ ID NO: 291), and

(SEQ ID NO:292) was identified as a non-binder. Variants based on binding analysis

((SEQ ID NO: 283), AB0053) and

Only ((SEQ ID NO:284), AB0085) was considered for mammalian generation and further characterization. Binding of AB0053 and AB0085 to hCLEC12A-His was characterized using surface plasmon resonance (SPR) at 37°C. Both mutations introduced to remove DS sequence liability affected binding to hCLEC12A-His (Table 61). Figure 69 shows FACS analysis of binding to hCLEC12A-his using a yeast library generated to remove sequence liability of CDRH3. This data indicated a complete loss of binding of the DS to DI engineered variant (AB0085). Introduction of DA instead of DS (AB0053) did not completely eliminate the binding, but caused clear heterogeneity in the binding sensorgrams. Following SPR analysis, the efficacy of the liability-repairing variants (AB0053 and AB0085) was tested in a KHYG-1-CD16aV-mediated cytotoxicity assay (Figure 70, Table 62). AB0085 lost the ability to kill AML cells as determined by a significant decrease in maximal killing rate. Although the EC50 of the AB0053 construct increased approximately 3-fold compared to the F3'-1602 TriNKET (AB0237) control, the rate of maximal killing was not affected. This finding is consistent with the 3-fold change in overall affinity for hCLEC12A established in SPR experiments (Table 61).

The binding affinity of F3'-1602 TriNKET to recombinant human CLEC12A was measured by SPR at 37°C using a Biacore 8K instrument. Briefly, human Fc-specific antibodies were covalently immobilized onto the carboxymethyldextran matrix of a CM5 biosensor chip at a density of approximately 8000-10000 resonance units (RU) by amine coupling chemistry to generate anti-hFc An IgG chip was made. The F3′-1602 TriNKET sample was captured on an anti-hFc IgG chip at a concentration of 1.5 μg/mL at a flow rate of 10 μL/min for 60 seconds to achieve approximately 150-250 RU capture levels. hCLEC12A-His was serially diluted 3-fold in HBS-EP+ buffer (1X; 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20 pH 7.4) containing 0.1 mg/mL bovine serum albumin (BSA). Dilutions (100 nM to 0.046 nM) were injected over the captured test substances at a flow rate of 30 μl/min. Association was monitored for 300 seconds and dissociation for 600 seconds. The surface was regenerated between cycles by injecting three pulses of 10 mM glycine-HCl, pH 1.7 at 100 μL/min for 20 seconds. HBS-EP+ (1X) containing 0.1 mg/mL BSA buffer was used throughout the experiment. Data were analyzed using Biacore 8K Insight Evaluation software (GE Healthcare).

Since the library approach yielded AB0053 as the only hit, which showed significantly different kinetics of binding to hCLEC12A than F3'-1602 TriNKET and inferior potency in cytotoxicity assays, the DS motif was It was concluded that it is necessary to maintain the structure of CDRH3 and therefore cannot be effectively removed. Instead, DS liability was controlled by a formula, as described in Example 14.

Example 14. Formulation of F3'-1602 In Example 13, the sequence liability site was found to be important for the ability of F3'-1602 to bind to CLEC12A and therefore cannot be removed. This example was designed to evaluate formulations that could maintain the stability of F3'-1602 despite the presence of sequence liability.

Briefly, the following three formulations were tested for F3'-1602.
(1) 20 mM histidine, 250 mM sucrose, and 0.01% polysorbate 80, pH 6.0 (referred to herein as "HST formulation"). F3'-1602 was diafiltered into HST buffer and the collected sample passed through a 0.2 μm filter. The final concentration of F3'-1602 was 19.7 mg/mL as measured by absorbance (A280) at 280 nM.
(2) 20 mM potassium phosphate, 10 mM sodium citrate, and 125 mM sodium chloride, pH 7.8 (referred to herein as a "phosphate/citrate/NaCl formulation"); F3′-1602 was buffer-switched to phosphate/citrate/NaCl formulation buffer using an Amicon Ultra 0.5 mL 30K device and Zeba Desalting Columns, and collected samples were passed through a 0.2 μm filter. passed through. The final concentration of F3'-1602 was 20.6 mg/mL as measured by A280.
(3) 20 mM citrate, 250 mM sucrose, and 0.01% polysorbate 80, pH 7.0 (referred to herein as "CST formulation"). F3'-1602 was diafiltered into CST formulation buffer and the collected sample passed through a 0.2 μm filter. The final concentration of F3'-1602 was 20 mg/mL as measured by A280.

Stability of F3'-1602 in HST formulations Accelerated stability studies were performed in HST buffer at 40°C for 4 weeks. Size exclusion chromatography (SEC) was used to monitor the formation of high molecular weight species (HMWS) and low molecular weight species (LMWS) as a function of storage conditions. Briefly, 50 μg of test substance was injected onto an Agilent 1260 Infinity II High Pressure Liquid Chromatography (HPLC) instrument equipped with a 1260 Quat Pump, 1260 Vialsampler, 1260 VWD. The sample was separated on a Tosoh TSKgel G3000SWxl, 7.8 mm x 30 cm id, 5 μm column. SEC running buffer was PBS, pH 7.0, 0.50 ml/min flow. Absorbance was monitored at both 214 nm and 280 nm and peak areas were manually integrated and reported as HMWS, LMWS, and percent monomer.

At the start of the study, F3'-1602 was analyzed by SEC and was 99.6% monomeric. After 4 weeks at 40°C the monomer was reduced to 93.1%. LMWS was not detected in controls and increased to 5.4% after 4 weeks at 40°C. HMWS was present in controls at 0.4% and increased to 1.5% after 4 weeks at 40°C (Table 63). These data suggest that F3'-1602 is mainly degraded by the formation of LMW species (fragmentation).

Binding affinities of F3'-1602 samples to recombinant human CLEC12A were measured by SPR at 37°C using a Biacore 8K instrument. Briefly, human Fc-specific antibodies were covalently immobilized onto the carboxymethyldextran matrix of a CM5 biosensor chip via amine coupling chemistry at a density of approximately 8000-10000 resonance units (RU). , produced an anti-hFc IgG chip. F3'-1602 samples were captured on anti-hFc IgG chips at a concentration of 1.5 μg/mL at a flow rate of 10 μL/min for 60 seconds to achieve a capture level of approximately 150-250 RU. hCLEC12A-His was serially diluted (100 nM to 0.046 nM) in 3-fold dilutions in HBS-EP+ (1X) containing 0.1 mg/mL bovine serum albumin (BSA) buffer and captured studies A flow rate of 30 μl/min was injected over the material. Association was monitored for 300 seconds and dissociation for 600 seconds. The surface was regenerated between cycles by injecting three pulses of 10 mM glycine-HCl, pH 1.7 at 100 μL/min for 20 seconds. HBS-EP+ (1X) containing 0.1 mg/mL BSA buffer was used throughout the experiment. Data were analyzed using Biacore 8K Insight Evaluation software (GE Healthcare). Kinetic rate constants and overall affinities were obtained using a 1:1 kinetic fitted binding model.

The binding of F3'-1602 to human CLEC12A decreased after 4 weeks of incubation at 40°C in HST formulations (Fig. 71). Differences in association and dissociation rate constants between control and 40° C. stressed samples are within the experimental variability of the assay (Table 64); Responses were reduced by more than 5-fold after incubation at 40° C. (FIG. 71). Nominal capture of F3'-1602 protein was the same in both stressed and control samples. Under these circumstances, R _max is approximately proportional to the active concentration of F3′-1602 captured on the Biacore chip. This result in turn indicates that more than 80% of F3'-1602 molecules lost the ability to bind CLEC12A after heat stress of HST formulations (Table 64).

Binding affinities of F3'-1602 samples to recombinant human NKG2D were measured by SPR at 37°C using a Biacore 8K instrument. Briefly, mFc-specific antibodies were covalently immobilized via amine coupling chemistry onto the carboxymethyldextran matrix of CM5 biosensor chips at densities of approximately 8000-10000 RU to generate anti-mFc IgG chips. mFc-tagged human NKG2D was captured at a concentration of 0.2 μg/mL onto an anti-mFc IgG chip at a flow rate of 10 μL/min for 60 seconds to achieve a capture level of approximately 35 RU. F3'-1602 was buffer exchanged into HBS-EP+ (1X) buffer and serially diluted (5000 nM to 9.78 nM) in 2-fold dilutions with HBS-EP+. The analyte (F3'-1602) was injected over the captured test material at a flow rate of 30 μl/min. Association was monitored for 60 seconds and dissociation was monitored for 60 seconds. The surface was regenerated between cycles by injecting 3 pulses of 10 mM glycine-HCl, pH 1.7 at 100 μL/min for 20 seconds. HBS-EP+(1X) buffer was used throughout the experiment. Data were analyzed using Biacore 8K Insight evaluation software (GE Healthcare). Binding affinities and kinetic rates were obtained using both 1:1 kinetic and steady-state affinity model fits.

After 4 weeks of incubation at 40° C. in HST formulations, the binding of F3′-1602 to human NKG2D was unchanged (FIG. 72, Table 65). Differences in association and dissociation rate constants between control and 40° C. stress samples, as well as overall affinities for NKG2D, were within the experimental variability of the assay. The maximal binding response did not change significantly. The results indicate that F3'-1602 maintained its ability to bind NKG2D under these heat stress conditions.

Binding affinities of F3'-1602 samples to biotinylated recombinant CD16a V158 (FCγRIIIa V158) receptors were measured by SPR. Briefly, FcγRIIIa V158 was captured on SA series S Biacore chips prior to kinetics evaluation experiments, achieving approximately 10-25 RU of capture. Trastuzumab, a well-characterized IgG1 bioagent, was used as an experimental control. This evaluation used injections of varying concentrations of ligand (F3'-1602 or trastuzumab) titrated with captured receptor (1500 nM to 11.7 nM and 0 nM in 1:1 serial dilutions). Each injection cycle consisted of association, dissociation, and regeneration steps. Association consisted of a 150 second ligand injection, dissociation was monitored for 300 seconds, followed by a 5 second injection of 2 mM sodium hydroxide to regenerate the surface between cycles. All steps were performed at a flow rate of 30 μL/min. 1×HBS-EP+ buffer was used as running/dilution buffer. Data were analyzed using Biacore 8K Insight evaluation software (GE Healthcare). This data was fitted with a 1:1 reaction kinetic model.

At 20 mg/mL, the binding of F3'-1602 to human CD16a V158 was unchanged after 4 weeks of incubation in HST formulations at 40°C (Figure 73 and Table 66). Differences in binding affinities and maximum binding responses (R _max ) between control and 40° C. stressed samples were within the experimental variation of the assay. This result indicates that F3'-1602 retained affinity for human CD16a under these accelerated storage conditions.

The effect of accelerated stability on F3'-1602 function was also evaluated in a cytotoxicity assay using KHYG-1-CD16a cells engineered to express CD16a in addition to NKG2D. CLEC12A ⁺ human cancer cell line (PL-21) was labeled with BATDA reagent. After labeling, cells were washed and resuspended in primary cell culture medium. BATDA-labeled cells, F3'-1602, and resting KHYG-1-CD16V cells were added to wells of a 96-well plate. Additional wells were prepared for maximal lysis of target cells by the addition of 1% Triton®-X. Spontaneous release from wells containing only BATDA-labeled cells was monitored. After 3 hours of culture, cells were pelleted, culture supernatants were transferred to clean microplates, and room temperature europium solution was added to each well. The plate was protected from light and incubated on a plate shaker at 250 rpm for 15 minutes. Plates were read using a SpectraMax i3X instrument. Specific lysis % was calculated as follows:
% specific lysis=((experimental release-spontaneous release)/(maximum release-spontaneous release)) ^* 100%

As shown in FIG. 74 and Table 67, the ability of F3′-1602 to kill PL21 CLEC12A ⁺ cancer cells decreased approximately 7-fold after 4 weeks at 40° C. for HST formulations, although the % maximal lysis decreased was negligible (5%).

A significant loss of binding to hCLEC12A and a corresponding loss of potency after incubation at 40° C. investigated the root cause of this behavior. UHPLC-MS/MS peptide mapping revealed that the aspartic acid in the anti-CLEC12A scFv (CDR-H3) was isomerized (Figure 75).

The Chothia-defined F3'-1602 CDRH3 contains four aspartic acids (DYDDSLDY [SEQ ID NO:293]). MS/MS was used to monitor the succinimide intermediates to determine which of them had undergone isomerization. Conversion to isoaspartic acid results in no mass difference, but the succinimide intermediate corresponds to the loss of water (approximately 18 Da). This mass shift can be used to identify isomerizing aspartic acids. MS/MS spectra of the unmodified chymotryptic peptide and the corresponding succinimide variants showed that the aspartic acid (DYDDSLDY [SEQ ID NO:293]) at position H99 (Chothia) of the scFv VH was responsible for the isomerization.

Stability of F3′-1602 in Phosphate/Citrate/NaCl Formulations Attempts to remove the liability by mutagenesis showed that the aspartic acid at position H99 of the CLEC12A-binding scFv was a key determinant of CDR structure, It became clear that it could not be easily replaced without losing binding to the target. Therefore, as the isomerization rate is pH dependent, limited pre-formulation development was performed to identify formulations with a more basic pH. The pH dependence was confirmed by highlighting F3'-1602 in citrate and phosphate buffers covering pH 5.5-7.5. After 4 weeks at 40° C. in phosphate buffer (pH 6.5/7.0/7.5), F3′-1602 showed a pH-dependent loss of binding to CLEC12A. Interestingly, no trends were observed in citrate buffers (pH 5.5/6.0/6.5). Based on these results, additional formulations were designed and common excipients (NaCl, sucrose, EDTA) were screened for their ability to stabilize CLEC12A binding (Table 68). This screen showed that high pH and NaCl have a protective effect on CLEC12A binding.

Based on these results, F3'-1602 is formulated in 20 mM potassium phosphate, 10 mM sodium citrate, 125 mM sodium chloride, pH 7.8 (phosphate/citrate/NaCl formulation).

Formation of HMWS and LMWS was assessed by SEC as described in the subsection "Stability of F3'-1602 in HST Formulations."F3'-1602 was incubated at 40°C for 4 weeks. At the start of the study, F3'-1602 was analyzed by SEC and was 97.9% monomeric. After 4 weeks at 40°C the monomer was reduced to 91.4%. LMWS increased from 1.8% to 5.0% after 4 weeks at 40°C. HMWS was present at 0.3% in controls and increased to 3.6% after 4 weeks at 40°C (Table 69). These data suggest that F3'-1602 was degraded by both aggregation (HMWS) and fragmentation (LMWS) in phosphate/citrate/NaCl formulations.

Fragmentation and aggregation of intact F3'-1602 were assessed in accelerated stability studies by non-reducing sodium dodecyl sulfate capillary electrophoresis (SDS-CE). Briefly, F3'-1602 was diluted to 0.5 mg/mL using 1X sample buffer to achieve a final sample volume of 50 μL. Internal standards and iodoacetamide were added to the samples, which were then incubated in a heat block at 70°C for 10 minutes, followed by an ice bath for 5 minutes. Samples were transferred to 96-well plates, centrifuged and loaded onto the instrument. Individual samples were loaded into capillaries at 4600 volts for 40 seconds and separated with a Maurice (ProteinSimple, San Jose, Calif.) at 5750 volts for 35 minutes.

The purity of the major peak of F3'-1602 decreased from 97.0% to 91.2% by the end of 4 weeks of incubation. LMWS increased correspondingly from 3.0% to 8.4% (Table 70). HMWS increased slightly (from non-detectable to 0.4%) during the study period as determined by non-reducing SDS-CE. Differences in the migration times of the main peaks of the overlay were within the experimental variability of this assay.

Fragmentation of the three individual polypeptide chains of F3'-1602 was assessed at each time point of the accelerated stability study by reducing SDS-CE. Purity was determined by the sum of the three strands in this assay. During the course of the study, the purity decreased from 97.4% to 95.2% (Table 71). This decrease in purity is the result of increased LMWS and is consistent with the SEC and non-reduced SDS-CE results. Differences in the migration times of the main peaks of the overlay were within the experimental variability of this assay.

Binding affinities of F3'-1602 samples to recombinant human CLEC12A were measured by SPR as described in the "Stability of F3'-1602 in HST Formulations" subsection. After 4 weeks of incubation in phosphate/citrate/NaCl formulations, human CLEC12A binding was somewhat reduced (Figure 76). Differences in association and dissociation rate constants between control and 40° C. stress samples were within the experimental variability of the assay (Table 72), but maximum binding responses (Rmax) adjusted for differences in capture were , decreased by about 10% after incubation at 40°C. Such a loss was well below the 80% observed after 4 weeks of incubation with HST formulations, and the phosphate/citrate/NaCl formulation reduced F3'-1602 under accelerated (40°C) conditions. It is shown that it could be stabilized. Therefore, isomerization of H99 was greatly reduced by formulation development, as explained in the subsection "Stability of F3'-1602 in HST Formulations."

Binding affinities of F3′-1602 samples to recombinant human NKG2D were measured by SPR as described in the subsection “Stability of F3′-1602 in HST Formulations”. The binding of F3′-1602 to human NKG2D was unchanged after 4 weeks of incubation at 20 mg/mL at 40° C. in a phosphate/citrate/NaCl formulation (FIG. 77 and Table 73). Differences in equilibrium binding affinities and Rmax between control and 40° C. stressed samples were within the experimental variation of the assay. This indicates that F3'-1602 retained affinity for human NKG2D under these accelerated storage conditions.

The binding affinity of F3′-1602 samples to biotinylated recombinant CD16a V158 (FCγRIIIaV158) receptor was determined by SPR, as described in the subsection “Stability of F3′-1602 in HST Formulations”. It was measured. F3′-1602 binding to human CD16a V158 was unchanged after 4 weeks of incubation at 20 mg/mL at 40° C. in a phosphate/citrate/NaCl formulation (FIG. 78 and Table 74). . Differences in binding affinities between control and 40° C. stress samples, and maximum binding responses were within the experimental variation of the assay. This indicates that F3'-1602 retains affinity for human NKG2D under these accelerated storage conditions.

As explained in the subsection "Stability of F3'-1602 in HST Formulations", the effect of accelerated stability on F3'-1602 function may be observed in KHYG-1-CD16a cells expressing NKG2D and CD16a. was also evaluated in a cytotoxicity assay using The ability of F3′-1602 to kill PL-21 CLEC12A+ cancer cells was unchanged after 4 weeks of incubation at 40° C. at a concentration of 20 mg/mL in a phosphate/citrate/NaCl formulation. (Figure 79 and Table 75). This result is in stark contrast to that observed after incubation with HST formulations, where F3'-1602 was stabilized at 20 mM phosphate, 10 mM citrate, 125 mM NaCl, pH 7.8. It is shown that.

Stability of F3′-1602 in CST Formulations Based on the stability data in phosphate/citrate/NaCl formulations, we conclude that loss of activity at 40° C. can be minimized in liquid formulations. was taken. The stability of F3'-1602 was further analyzed in a lyophilization compatible liquid formulation containing sucrose as a cryoprotectant.

Accelerated stability studies at 20 mg/mL were 20 mM citrate, 250 mM sucrose, 0.01% Tween®-80, pH 7.0 (CST formulation), refrigerated (2-8°C). and accelerated (25 and 40° C.) conditions for 4 weeks. A series of assays were performed on samples stressed at 40°C, with binding and potency to CLEC12A (SPR) monitored in the 2-8°C and 25°C arms.

Formation of HMWS and LMWS was assessed by SEC as described in the "Stability of F3'-1602 in the HST formulation" subsection. At the start of this study, F3'-1602 was analyzed by SEC and was 99.6% monomeric. After 4 weeks at 40°C the monomer was reduced to 98.6%. LMWS was not detected in controls and increased to 0.5% after 4 weeks at 40°C. HMWS was present in controls at 0.4% and increased to 1.0% after 4 weeks at 40°C (Table 76). These data suggest that F3'-1602 was resistant to both aggregation and fragmentation under the accelerated conditions of the CST formulation.

Fragmentation of intact F3'-1602 is a sub-section of "Stability of F3'-1602 in the phosphate/citrate/NaCl formulation". Each time point was evaluated in an accelerated stability study with non-reducing SDS-CE as described in section. The purity of the F3'-1602 major peak decreased from 96.1% to 91.0% by the end of 4 weeks of incubation. LMWS increased correspondingly from 3.9% to 9.0% (Table 77). There was no increase in HMWS over the study period as determined by non-reducing SDS-CE. Differences in the migration times of the main peaks of the overlay were within the experimental variability of this assay.

Fragmentation of the three individual polypeptide chains of F3′-1602 is the “Stability of F3′-1602 in the phosphate/citrate/NaCl formulations. Each time point was assessed in an accelerated stability study with reducing SDS-CE as described in the NaCl formulation subsection. Purity was determined by the sum of the three strands in this assay. During the course of the study, the purity decreased from 98.3% to 97.7% (Table 78). Differences in the migration times of the main peaks of the overlay were within the experimental variability of this assay.

The binding affinity of F3'-1602 samples to recombinant human CLEC12A was determined as described in the subsection "Stability of F3'-1602 in the HST formulation". Measured by SPR. Binding of human CLEC12A decreased after 4 weeks of incubation at 20 mg/mL at 40° C. in CST formulations (FIG. 80 and Table 79). Differences in association and dissociation rate constants between control and 40° C. stressed samples are within the experimental variability of the assay, although the apparent binding response is approximately 15° C. after stress at 40° C. decreased by 17%. This indicates that 15-17% of F3'-1602 lost the ability to bind CLEC12A after 4 weeks under these heat stress conditions. Classical SPR binding assays are a good indicator of potential loss of activity when there is a significant difference in binding (20% or more), but not enough to address the effects of small changes in sample activity. Unreliable (especially at low replicate numbers); if necessary, other analytical methods are used to elucidate changes with greater scientific certainty.

At lower temperatures (refrigerated and 25° C.), no apparent binding adjusted for differences in affinity or capture levels for hCLEC12A was observed after 4 weeks in cST formulations (FIGS. 81, Tables 80, and 81). ).

Binding affinities of F3′-1602 samples to recombinant human NKG2D were measured by SPR as described in the subsection “Stability of F3′-1602 in HST Formulations”. F3′-1602 binding to human NKG2D was unchanged after 4 weeks of incubation at 20 mg/mL at 40° C. in CST formulations (FIG. 82 and Table 82). Differences in equilibrium binding affinities and apparent Rmax between control and 40° C. stressed samples were within the experimental variation of the assay. This indicates that F3'-1602 retained affinity for human NKG2D under these accelerated storage conditions.

The binding affinity of F3′-1602 samples to biotinylated recombinant CD16a V158 (FCγRIIIaV158) receptors was determined by SPR, as described in the subsection “Stability of F3′-1602 in HST Formulations”. It was measured. F3′-1602 binding to human CD16a V158 was unchanged after 4 weeks of incubation at 20 mg/mL at 40° C. in CST formulations (FIG. 83 and Table 83). Differences in equilibrium binding affinities and apparent R _max between control and 40° C. stressed samples are within the experimental variation of the assay. This indicates that F3'-1602 retained affinity for human CD16a under these accelerated storage conditions.

As explained in the subsection "Stability of F3'-1602 in HST Formulations", the effect of accelerated stability on F3'-1602 function may be observed in KHYG-1-CD16a cells expressing NKG2D and CD16a. was also evaluated in a cytotoxicity assay using The ability of F3′-1602 to kill PL-21 CLEC12A+ cancer cells was unchanged after 4 weeks of incubation at 40° C. at a concentration of 20 mg/mL in CST formulation (FIG. 84 and Table 84).

Example 15. Manufacturability of F3'-1602 in CST Formulations Various conditions during manufacturing of F3'-1602 can affect its stability. This example was designed to assess the stability of F3'-1602 in CST formulations through freeze/thaw, agitation, low and high pH, forced oxidation, and low pH holding. Methods for measuring protein integrity and function, as provided in Example 14, are not repeated in this example.

Freeze/Thaw Stability during freeze/thaw cycles is important for biotherapy, as process intermediates and bulk drug substances can be frozen to ensure stability between process steps. To create freeze/thaw stress, 8.0 mg F3'-1602 was added to 20 mM sodium citrate, 250 mM sucrose, 0.01% Tween®-80, pH 7 using an Amicon Ultra 15 ml 30K device. 0.0 and made up to 400 μL with the same buffer (approximately 20 mg/mL F3′-1602, concentration confirmed by A280 using a Nanodrop One spectrophotometer). The concentrated sample was divided into 4 x 100 μL aliquots. Controls were immediately stored at -80°C. The remaining three aliquots are subjected to freeze/thaw cycles at −80° C. in Styrofoam containers to slow down the freezing process. At cycles 2, 4, and 6, aliquots were removed and transferred to −80° C. storage until analysis.

Freeze/thaw stability of F3'-1602 was assessed by measuring protein concentration by A280 at the completion of the study. The concentration of F3′-1602 was 20.4 mg/mL in the control and 20.2 mg/mL after 6 freeze/thaw cycles with no allele loss due to freeze/thaw stress. is shown.

To assess whether F3'-1602 is stable through freeze/thaw cycles, the test article was frozen and thawed six times and analyzed by SEC after cycles 2, 4, and 6. F3'-1602 at Time 0 was 99.5% monomeric and the % monomer did not change through all six freeze/thaw cycles (Table 85). This result indicates that F3'-1602 was resistant to both aggregation and fragmentation during freeze/thaw stress.

Fragmentation of intact F3'-1602 was assessed in a freeze/thaw stability study with non-reducing SDS-CE. The major peak purity of F3'-1602 decreased minimally (96.1% to 95.5%) after 6 freeze/thaw cycles. LMWS increased correspondingly from 3.9% to 4.5% (Table 86). No HMWS was detected during the study period. These results show that F3'-1602 is resistant to fragmentation at 20 mM citrate, 250 mM sucrose, 0.01% Tween®-80, pH 7.0. Differences in the migration times of the main peaks of the overlay were within the experimental variability of this assay.

Fragmentation of the three individual polypeptide chains of F3'-1602 was assessed in a freeze/thaw stability study by reducing SDS-CE. Purity was determined by the sum of the three strands in this assay. The purity of F3'-1602 did not change after 6 freeze/thaw cycles (Table 87). These results indicate that F3'-1602 was resistant to fragmentation with 20 mM citrate, 250 mM sucrose, 0.01% Tween®-80, pH 7.0. Differences in the migration times of the main peaks of the overlay were within the experimental variability of this assay.

Agitation Stability during agitation is important for biotherapeutics because process intermediates and bulk drug substances can be subjected to shear stress during processing steps. To create agitation stress, 8.0 mg F3′-1602 was added to 20 mM sodium citrate, 250 mM sucrose, 0.01% Tween®-80, pH 7.0 using an Amicon Ultra 15 mL 30K device. and brought to 400 μL with the same buffer (approximately 20 mg/mL F3′-1602, concentration confirmed by A280 using a Nanodrop One spectrophotometer). The concentrated sample was transferred to a glass vial with a stir bar, placed on a stir plate and stirred at 60 rpm, 4° C. for 18 hours. After stirring, the samples were stored at -80°C until analysis.

The agitation stability of F3'-1602 was evaluated. After stirring, there was a visible protein precipitate. The sample was spun down and the supernatant used in all characterization assays. Protein concentration in the supernatant was assessed by A280. The concentration of F3'-1602 was 20.4 mg/mL in control and 16.8 mg/mL after agitation stress.

To determine whether F3'-1602 is stable during agitation stress, test substances were analyzed by SEC before and after agitation. The F3'-1602 control was 99.5% monomer and the % monomer did not change after agitation (Table 88). This indicates that F3'-1602 was resistant to both aggregation and fragmentation during agitation.

Fragmentation of intact F3'-1602 was assessed after agitation with non-reducing SDS-CE. The major peak purity of F3′-1602 decreased minimally (96.1% to 95.5%) after stirring. LMWS increased correspondingly from 3.9% to 4.5% (Table 89). No HMWS was detected over the study period. These results indicate that F3'-1602 was resistant to fragmentation with 20 mM citrate, 250 mM sucrose, 0.01% Tween®-80, pH 7.0. Differences in the migration times of the main peaks of the overlay were within the experimental variability of this assay.

Fragmentation of the three individual polypeptide chains of F3'-1602 was assessed in agitation studies with reducing SDS-CE. Purity was determined by the sum of the three strands in this assay. The purity of F3'-1602 did not change after agitation stress (Table 90). These results indicate that F3'-1602 was resistant to fragmentation with 20 mM citrate, 250 mM sucrose, 0.01% Tween®-80, pH 7.0. Differences in the migration times of the main peaks of the overlay were within the experimental variability of this assay.

low pH and high pH
To create a low pH stress, 1.0 mg of F3′-1602 was buffer switched to 20 mM sodium acetate, pH 5.0 using an Amicon Ultra 0.5 mL 10K device and diluted to 1.0 mL with the same buffer. (approximately 1 mg/mL F3′-1602, concentration confirmed by A280 using a Nanodrop One spectrophotometer). Samples were split. Half was stored at -80°C as a control and the other half was incubated at 40°C for 2 weeks. After stirring, the samples were stored at -80°C until analysis.

To generate high pH stress, 1.0 mg of F3'-1602 was buffer switched to 20 mM Tris, pH 8.3 using an Amicon Ultra 0.5 mL 10K device and made up to 1.0 mL with the same buffer. (Approximately 1 mg/mL F3'-1602, concentration confirmed by Nanodrop One A280). Samples were split. Half was stored at -80°C as a control and the other half was incubated at 40°C for 2 weeks. After stirring, the samples were stored at -80°C until analysis. The pKa of Tris changes as a result of temperature. At 40° C., the pH 8.3 buffer was approximately pH 8.0.

To assess the chemical stability of F3'-1602, the test substances were kept at pH 5.0 and pH 8.0 for two weeks at 40°C. At low pH, typical chemical modifications are isomerization and fragmentation of aspartic acid, whereas at high pH the major degradation pathways are deamidation and oxidation. To confirm the integrity of F3'-1602 after incubation, control and stress samples were analyzed by SEC (Table 91). The results show that F3'-1602 remained mostly intact with low levels of fragmentation, as expected under these harsh conditions.

Chemical modifications of amino acid side chains can be routinely observed on a global scale using capillary isoelectric focusing (cIEF). To assess modification of F3′-1602 samples maintained at low and high pH, F3′-1602 was diluted to 1 mg/mL using MilliQ water and 15 μL of sample was added to 60 μL of master mix. (water, methylcellulose, Pharmalyte 3-10, arginine, pI markers 4.05 and 9.99), vortexed and centrifuged briefly. 60 μL of sample was aspirated from the top of the solution, added to a 96-well plate and centrifuged prior to testing. During the experiment, the samples were separated with a Maurice (ProteinSimple, San Jose, Calif.) at 1500 volts for 1 minute followed by 3000 volts for 8 minutes.

The cIEF profile of F3'-1602 after 2 weeks of holding at pH 5.0 showed an increase in basic species from 8.9 to 39.1% (Figure 85 and Table 92). A similar increase in acidic species was detected by cIEF after holding F3'-1602 at pH 8.0. A corresponding decrease in major peak intensity (51.4-29.0%) was also observed (Figure 86 and Table 92). Without wishing to be bound by theory, it is possible that deamidation of the entire F3'-1602 sequence leads to this acidic shift.

To determine whether the pH stress-induced changes observed by cIEF are due to alterations in complementarity determining regions (CDRs), the same pH 5 and pH 8 stress samples were analyzed by LC-MS/MS peptide maps. analyzed. Briefly, 50 μg of F3′-1602 was diluted with 6 M guanidine hydrochloride in 250 mM Tris, pH 8.5 and disulfide bonds were reduced with 25 mM dithiothreitol at 40° C. in a thermomixer for 30 minutes. The reduced cysteines were then alkylated with 50 mM iodoacetamide for 60 minutes at room temperature in the dark. Reduced and alkylated proteins were buffer switched to 50 mM Tris, pH 8.0 and digested using trypsin (1:25 enzyme:substrate) for 1 hour at 37°C. LC-MS/MS was run on a Thermo Vanquish UHPLC coupled to a QExactive+ mass spectrometer. Briefly, 5 μg of protein digest was separated on a Waters UPLC BEH Peptide C18 column (2.1×150 mM) over 150 minutes using a 2%-40% acetonitrile gradient at 0.3 mL/min. Mass spectra were acquired from 230-2000 m/z in positive mode at a resolution of 35,000 for full MS scans and 17,500 for Top 10 MS/MS scans. UPLC-MS/MS files were analyzed using the Byos PTM workflow (Protein Metrics). Peptides were identified using accurate mass and MS/MS (searching F3'-1602 sequence).

Based on peptide map data, all but one of the CDRs of F3'-1602 were resistant to modification during low and high pH stress. Only CDRH3 of CLEC12A-binding scFv-Fc chains showed evidence of modification (isomerization) after pH 5 stress (Table 93). This modification is the same as identified in the accelerated (40°C) stability study.

Binding affinities of F3′-1602 samples to recombinant human CLEC12A were measured by SPR as described in Example 14. After 2 weeks of incubation at 40° C. in 20 mM sodium acetate, pH 5.0, the binding of F3′-1602 to human CLEC12A was reduced (FIG. 87 and Table 94). Although the differences in association and dissociation rate constants between control and low pH 40° C. stress samples were within the experimental variation of the assay, the apparent maximal binding response of the stress samples was higher than that of the pH 5 post-stress control. was about half of This indicates that F3'-1602 has a reduced ability to bind CLEC12A under these conditions. After 2 weeks of incubation in 20 mM Tris, pH 8.0 at 40° C., the binding of F3′-1602 to human CLEC12A was unchanged (FIG. 88 and Table 94). This indicates that F3'-1602 retained affinity for CLEC12A under these accelerated pH stress conditions.

Binding affinities of F3′-1602 samples to recombinant human NKG2D were measured by SPR as described in Example 14. After 2 weeks of incubation at 40° C. in 20 mM sodium acetate, pH 5.0, the binding of F3′-1602 to human NKG2D was unchanged (FIG. 89 and Table 95). This indicates that F3'-1602 retained affinity for NKG2D under these accelerated pH stress conditions. After 2 weeks of incubation in 20 mM Tris, pH 8.0 at 40° C., the binding of F3′-1602 to human NKG2D was unchanged (FIG. 90 and Table 95). This indicates that F3'-1602 retained affinity for NKG2D under these accelerated pH stress conditions.

The effects of low and high pH stress on F3'-1602 function were assessed in a cytotoxicity assay using KHYG-1-CD16a cells expressing NKG2D and CD16a, as described in Example 14. The potency of F3′-1602 against PL-21 CLEC12A+ cancer cells, as reflected by the EC50 and maximal lysis after pH 5.0 and 8.0 stress, decreased approximately 2-fold after pH 5 stress (FIG. 91 and table 96) and did not change after pH 8 stress (Figure 92 and Table 96). Without wishing to be bound by theory, it is possible that the loss of potency after pH 5 stress was due to the same isomerization of the scFvCDR-H3 aspartate identified in the initial accelerated stability study. be. The difference in pH 5 and pH 8 results further indicates that F3'-1602 was more stable at higher pH.

Forced Oxidation One of the most common stability indicators of protein modification is the oxidation of methionine residues. To identify oxidation hotspots, 1.0 mg of F3'-1602 was diluted to 1 mg/mL using phosphate buffered saline (PBS). 3.3 μL of 3% hydrogen peroxide was added to a 0.5 mL aliquot of F3'-1602 (0.02% hydrogen peroxide). Oxidation of F3'-1602 was allowed to proceed for 24 hours at room temperature in the dark. After 24 hours, samples were buffer switched to PBS, pH 7.4 using an Amicon Ultra 0.5 mL 10K MWCO device according to the manufacturer's instructions. A non-oxidized control (0.5 mL) was stored at -80°C.

There were no significant differences in % monomer, HMWS or LMWS in F3′-1602 after forced oxidation (Table 97).

After forced oxidation, the purity of F3'-1602 by non-reducing SDS-CE decreased to less than 1% (Table 98). This indicates that F3'-1602 was resistant to fragmentation during forced oxidation. No new fragments were detected after forced oxidation. The peak shape of the oxidized samples changed after oxidative stress. Without wishing to be bound by theory, this suggests that there was no longer a homogeneous population and that high levels of oxidation resulted in conformational changes that were not present in the control sample of F3'-1602. can show Differences in the migration times of the main peaks of the overlay were within the experimental variability of this assay.

After forced oxidation, the purity of F3'-1602 by reducing SDS-CE did not change (Table 99). This indicates that F3'-1602 was resistant to fragmentation during forced oxidation. No new fragments were detected after forced oxidation. Differences in the migration times of the main peaks of the overlay were within the experimental variability of this assay.

In addition to determining the biophysical effects of forced oxidation by SEC, it is important to understand which amino acids are most susceptible to oxidation. There are no methionine residues in the F3'-1602 CDR, but several methionines are found in the framework and constant domains. Oxidation levels were low at all sites in the control, and only two sites showed a significant increase in oxidation after exposure to hydrogen peroxide (Table 100). Low levels of oxidation have been observed with other therapeutic mAbs.

The binding affinity of forced oxidative stress F3'-1602 samples to recombinant human CLEC12A was measured by SPR as described in Example 14. After incubation for 24 hours at room temperature in the presence of 0.02% hydrogen peroxide, the kinetics and affinity of F3′-1602 binding to human CLEC12A and the apparent R _max were unchanged (FIG. 93). and Table 101). This indicated that F3'-1602 retained binding to CLEC12A after forced oxidation and modification of methionines in the constant region and framework, but did not affect the affinity of F3'-1602 for hCLEC12A. ing.

The binding affinity of forced oxidative stress F3'-1602 samples to recombinant human NKG2D was measured by SPR as described in Example 14. After incubation for 24 hours at room temperature in the presence of 0.02% hydrogen peroxide, the kinetics and affinity of F3'-1602 binding to human NKG2D did not change (Figure 94 and Table 102). This indicates that F3'-1602 retains binding to NKG2D after forced oxidation and modification of methionines in the constant region and framework without affecting the affinity of F3'-1602 for hNKG2D. .

The effect of forced oxidative stress on F3′-1602 function was assessed in a cytotoxicity assay using KHYG-1-CD16a cells expressing NKG2D and CD16a, as described in Example 14. As shown in Figure 95 and Table 103, the potency (EC50 and maximal lysis) of F3'-1602 did not change after oxidative stress.

Low pH Hold Virus inactivation by low pH hold is an important step in biologics manufacturing. To assess the stability of F3'-1602 at low pH holding, F3'-1602 was captured on a MabSelect Sure Protein A column and eluted with 100 mM glycine, pH 3.0. Peak fractions were pooled and 12 mL of protein A eluate was pH adjusted to pH 3.3 with additional 100 mM glycine pH 3.0 buffer. The pH adjusted eluate was kept at room temperature. After 1.5 hours, the eluate was brought to neutral pH using 1.0 M Tris-HCl, pH 8.3. The pH-kept sample was further purified by cation exchange chromatography. The final product was buffer exchanged into PBS pH 7.4 for analysis.

After incubation at low pH, F3'-1602 was analyzed by SEC to determine whether retention caused aggregation. There was no increase in HMW and LMW content in F3'-1602 due to the low pH holding step (Table 104).

The purity of F3'-1602 by non-reducing SDS-CE did not change after low pH hold (Table 105). This indicates that F3'-1602 was resistant to fragmentation during low pH holding. No new fragments were detected after the low pH hold. Differences in the migration times of the main peaks of the overlay were within the experimental variability of this assay.

Purity of F3'-1602 by reducing SDS-CE did not change after low pH hold (Table 106). This indicates that F3'-1602 was resistant to fragmentation during low pH holding. No new fragments were detected after the low pH hold.

Chemical modifications of amino acid side chains can usually be observed globally using cIEF. The cIEF profiles of the F3'-1602 control and the low pH hold lot were visually very similar, with relative quantification of acidic, major, and basic species all within 1% of each other. , indicating that low pH retention had no measurable effect on the charge profile of F3′-1602 (Table 107).

F3'-1602 samples were analyzed at intact mass to monitor modifications and fragmentation as a result of low pH holding. Observed masses in both the control and low pH hold samples were consistent with each other and with the theoretical F3'-1602 mass (G0F/G0F glycoform: 126,360.4 Da). Low pH retention did not affect the mass of F3'-1602 (Table 108) and no fragmentation was observed.

Low pH holding did not induce changes in the kinetics or affinity of F3'-1602 binding to human CLEC12A (Figure 96 and Table 109). Differences in association and dissociation rate constants, and apparent maximum binding responses (Rmax) between control and low pH holding samples were within the experimental variability of the assay. This indicates that F3'-1602 retained affinity for CLEC12A after the low pH holding step of biotherapeutic manufacture.

Human NKG2D binding was unchanged after low pH hold (Figure 97 and Table 110). Differences in steady state affinities and apparent maximal binding response affinities between control and 40° C. stressed samples were within the experimental variation of the assay. This indicates that F3'-1602 retained its affinity for human NKG2D after low pH holding.

After low pH holding, the binding of F3'-1602 to human CD16a V158 was unchanged (Figure 98 and Table 111). Differences in binding affinities and maximal binding responses between control and low pH holding samples were within the experimental variation of the assay. This indicates that F3'-1602 retained affinity for human CD16a under these accelerated storage conditions.

In addition to compromising biophysical stability and integrity, low pH can cause chemical degradation of amino acid side chains, especially isomerization of aspartic acid. Chemical degradation, if any, of F3'-1602 in regions critical for function should manifest as decreased potency in potency assays. Therefore, the efficacy of F3'-1602 against CLEC12A ⁺ PL-21 cancer cells was tested after low pH hold in the KHYG1-CD16V bioassay. The potency of F3'-1602 was not altered by low pH holding (Figure 99 and Table 112).

Colloidal Stability PEG precipitation was used to assess the colloidal stability of F3′-1602. Briefly, concentrated F3′-1602 (approximately 20 mg/mL) was diluted to 1 mg/mL in 100 μL aliquots (10 mM sodium acetate, pH 5.0) with a final concentration of PEG 6000 of 0-30. % (w/v) range. Aliquots were mixed thoroughly and left overnight at 2-8°C. The next day, the tubes were centrifuged to pellet the precipitate and the concentration of the supernatant was measured by A280 on a Lunatic (Unchained Labs).

The solubility of F3'-1602 in acetate buffer pH 5.0 at increasing concentrations of PEG 6000 (0%-30%) was monitored by A280. The midpoint transition (Cm) at 10 mM sodium acetate, pH 5.0 was 24.5%, indicating that F3'-1602 has high solubility in this buffer (Figure 100). As comparators, trastuzumab and adalimumab were also evaluated in a PEG precipitation assay with 10 mM sodium acetate, pH 5.0. The midpoint transitions for trastuzumab and adalimumab used as assay controls were >30% and 11.7% PEG-6000, respectively.

F3'-1602 was then concentrated using a 30 kDa molecular weight cutoff device in PBS, pH 7.4. Concentration was determined by A280 on a Nanodrop and product quality was monitored by SEC. Test substances were injected into an Agilent 1260 Infinity II high pressure liquid chromatography instrument equipped with a 1260 Quat Pump, 1260 Vialsampler, 1260 VWD. Samples were separated on a Superdex200 Increase 10/300 GL column. SEC running buffer was PBS, pH 7.4, flow rate 0.60 mL/min. Absorbance was monitored at both 214 nm and 280 nm and peak areas were manually integrated and reported as HMWS, LMWS, and percent monomer.

F3'-1602 was concentrated to approximately 25, 50, 100, and 150 mg/mL PBS, pH 7.4, and recovery was monitored by visual inspection, concentration (A280), and SEC. F3'-1602 could be concentrated to >150 mg/mL in PBS, pH 7.4, with minimal percent monomer loss and no visible precipitate (Figure 101 and Table 113).

Example 16. Stability and Manufacturability of F3′-1602 and AB0010 F3′-1602 and AB0010 are TriNKETs in F3′ and F3 formats, respectively, with CLEC12A binding sites derived from the same humanized anti-CLEC12A antibody. As described in Examples 5 or 6, these two TriNKETs have binding affinities similar to CLEC12A. This example was designed to compare the stability and manufacturability of F3'-1602 and AB0010.

Briefly, F3′-1602 and AB0010 were prepared in 20 mM sodium phosphate, 10 mM sodium citrate, 125 mM NaCl, and 0.01% PS-80 at pH 7.8 as described in Example 14. It was formulated with Formulated proteins were subjected to heat stress at 40° C. for 4 weeks and their stability was evaluated in non-reduced SDS-CE, reduced SDS-CE, SEC, and cytotoxicity assays as described in Example 14. rated by

The data presented in Table 114 indicate that the LMW fraction of stressed AB0010 samples was significantly increased over that of stressed F3'-1602 samples. The data presented in Table 115 indicate that the purity of the stressed AB0010 sample was significantly reduced from that of the stressed F3'-1602 sample. These results indicate that AB0010 deteriorated significantly more than F3'-1602 under heat stress conditions.

As shown in Table 116, the amount of HMWS in stressed AB0010 samples increased significantly over that in stressed F3'-1602 samples. This result suggests that AB0010 aggregated significantly more than F3'-1602 under heat stress conditions.

As described in Example 14, sucrose is more compatible with lyophilized presentation than NaCl. Therefore, F3'-1602 and AB0010 were also formulated in 20 mM sodium phosphate, 10 mM sodium citrate, 250 mM sucrose, and 0.01% polysorbate 80, pH 7.8. The TriNKET protein in each formulation was then subjected to heat stress at 40° C. for 4 weeks and its ability to mediate NK cell-mediated cytotoxicity was assessed by the cytotoxicity assay described in Example 14. As shown in Figures 102A and 102B and Table 117, the ability of F3'-1602 and AB0010 to mediate killing of PL21 CLEC12A ⁺ cancer cells by NKG2D- and CD16a-expressing KHYG-1-CD16a cells was enhanced by heat stress. was not significantly affected by

Example 17. Molecular format, design, structure and characteristics of CLEC12A TriNKET AB0089 Purpose The purpose of this study is to describe the molecular format, design, structure and properties of CLEC12A TriNKET AB0089. AB0089 is an improved version of AB0237 in which the primary sequence of the CLEC12A-binding VH is optimized to remove the liability of isomerization sequences while maintaining affinity for CLEC12A and potency for cytotoxic lysis of CLEC12A+ AML cells. there is In addition, we aimed to reduce the number of mouse backmutations in the framework regions that increased the humanness of the molecule. Specifically, in this report, we a) describe the molecular format and design of AB0089; b) review protein engineering efforts to remove sequence liability of CDRH3; c) mouse backmutation d) provides information on the expression and purification of AB0089; e) describes the basic biochemical and biophysical characterization of the molecule; f) Determine the affinity of AB0089 to recombinant human targets (CLEC12A, NKG2D and CD16a); g) demonstrate binding of AB0089 to AML cancer cell lines expressing human CLEC12A; h) demonstrate selectivity of AB0089; i) determine the potency of AB0089 in killing AML cancer cells; j) assess binding epitopes; k) assess binding to FcγRs and FcRs and compare to IgG1 mAb control; and l) accelerated stability, The behavior of AB0089 in developability studies such as forced disassembly and manufacturability is described.

タンパク質試薬

Study Design During the feasibility evaluation of the CLEC12A-specific AB0237 TriNKET, we identified the isomerization liability of CDRH3, leading to decreased binding to CLEC12A and potency under severe stress. Yeast display was used to remove sequence liabilities and optimize the CMC properties of the molecule. In addition, the framework of the molecule was further optimized to remove mouse backmutations and replace them with the corresponding human residues, thus improving the humanness of the molecule. The properties of AB0089 were assessed by binding to recombinant hCLEC12A, binding to isogenic and cancer cells expressing CLEC12A, and potency of the molecule against CLEC12A+ AML cancer cells. Biophysical and biochemical properties of AB0089 were determined, including thermal stability (DSC), hydrophobicity (HIC), and pI (cIEF). Molecular modeling was used to assess the distribution of surface charge and hydrophobic patches of AB0089 and benchmarked with a clinical stage monoclonal antibody. Specificity of binding was assessed by PSR, binding to cells not expressing the target of interest, and HuProt™ arrays. AB0089 was expressed in two different mammalian cell lines (Expi293 and ExpiCHO) and purified using the Dragonfly Tx2 step platform purification method. Binding of AB0089, including CLEC12A, NKG2D, CD16a (V158 and F158), an expanded panel of Fcγ receptors (CD32a (H131 and R131), CD16b, CD32b, cynomolgus CD16, human and cynomolgus CD64), and FcRn (human and cynomolgus) Properties were fully characterized. Finally, AB0089 was tested in platform formulations (20 mM histidine, 250 mM sucrose, 0.01% Tween®-80, pH 6.0) and 20 mM citrate, 250 mM sucrose, 0.01% Tween®- 80, accelerated (40°C) stability in pH 7.0 buffer, forced degradation (long-term pH 5, pH 8 stress, forced oxidation), and manufacturability (freeze/thaw, agitation, low pH hold), The entire development feasibility panel of assays was evaluated.
Materials and methods Materials Test substances

Protein reagent

Methods Cell Binding by Flow Cytometry Binding of AB0089 to isogenic CLEC12A-expressing cells and PL-21 cancer cells was performed as described in detail in Example 19. EC50 values were derived from cell binding curves using GraphPad Prism software (4-parameter logistic non-linear regression curve fitting model).

Yeast Library Construction and Evaluation A yeast display affinity library (designated Library 17) was constructed using a synthetic CDRH3 oligonucleotide pool using a walking mutagenesis strategy to generate AB0237 CDRH3 (YDYDDSLDY, SEQ ID NO: 139) were replaced with randomization of all 20 amino acids, either as single sites or in pairs (Figure 103).

First, synthetic scFv double-stranded DNA was designed by replacing CDRH3 with a unique BamH1 restriction site. The scFv was then cloned into the yeast display plasmid (pYES-Aga2 vector) by Hifi cloning method to obtain the pYES-Aga2-scFv-BamH1 plasmid. DNA oligos with regions of homology with the vector were XDYDDSLDY (SEQ ID NO: 305), YXYDDSLDY (SEQ ID NO: 306), YDXDDSLDY (SEQ ID NO: 307) by randomization introduced into the original CDRH3 (YDYDDSLDY, SEQ ID NO: 139). ), YDYXDSLDY (SEQ ID NO: 308), YDYDXSLDY (SEQ ID NO: 309), YDYDDXLDY (SEQ ID NO: 310), YDYDDSXDY (SEQ ID NO: 311), YDYDDSLXY (SEQ ID NO: 312), YDYDDSLDX (SEQ ID NO: 313), XXYDDSLDY (SEQ ID NO: 314) ( where X=any amino acid) and pooled together. The pYES-Aga2-scFv-BamHI vector was then digested with BamHI and the digested plasmid was electroporated with DNA oligo pools to obtain the completed yeast display library 17. Through the process of homologous recombination, yeast internally ligates the library oligos with the linearized plasmid, so that each yeast cell contains one plasmid with one CDRH3 mutation oligo. Following selection and characterization of library 17 output, a similar affinity maturation strategy targeting CDRH2 for walking mutagenesis in a single optimized CDRH3 background was employed. The library for this second round of affinity maturation is referred to herein as library 30.

Libraries were evaluated by FACS sorting after growth in D-UT medium. Twenty-four hours before sorting, yeast was added to a final concentration of 0.50. D. /ml (measured by visible light spectrophotometer), spun down, washed twice with GR-UT medium, and incubated overnight in the same medium. On the day of the experiment, 3 O.D. D. of cells were harvested, tested and placed in wells of a 96-well U-bottom plate. This was repeated until the number of antigens desired to be tested. Cells were then washed twice with PBS-F (PBS + 0.25% BSA) and then incubated with various concentrations of biotinylated CLEC12A-His for 30 minutes. Yeast cells were then stained with FITC-labeled anti-Flag and streptavidin PE and the highest affinity binders were sorted using BD FACSAria.

The final yeast library was analyzed by flow cytometry. Yeast libraries were initially plated on D-UT medium 24 hours prior to characterization. Twenty-four hours before flow cytometry, yeast was added to a final concentration of 0.50. D. /mL, spun down, washed twice with GR-UT medium, and incubated overnight in the same medium. 0.2 O.D. per antigen tested on the day of the experiment. D. cells were taken into each well of a 96-well U-bottom plate. The cells were washed with PBS-F (PBS + 0.25% BSA), then biotinylated CLEC12A-His was added to a final concentration of 10 nM for 30 minutes. The cells were washed again and non-biotinylated CLEC12A-His (1 μM final concentration) was added for an additional 40 minutes and samples were removed every 20 minutes. Anti-Flag-FITC and streptavidin PE were added, incubated in the dark on ice for 30 minutes, washed twice, resuspended in PBS-F and analyzed using a FACS Celesta.

Site-directed mutagenesis A gBlock (double-stranded DNA fragment) with a DS to DT mutation encoding the AB0089 scFv targeting the CLEC12A arm was ordered from Integrated DNA Technologies (Coralville, Iowa). It was cloned into the pcDNA3.4 vector according to the protocol described below.

Codon Optimization DNA codon optimization of the scFv portion of the S chain of AB0089 was performed via the Codon Optimization Tool of Integrated DNA Technologies (Coralville, Iowa), and the linker and Fc portion were obtained using GeneArt Gene Synthesis (Thermo Fisher Scientific, Waldorf). (As part of the vector backbone) was codon optimized using the Codon Optimization Tool from Co., MA, USA. DNA codon optimization for expression of AB0089 H and L chains was performed using the codon optimization tool of GeneArt Gene Synthesis (Thermo Fisher Scientific, Waltham, Mass., USA). The goal was to achieve high expression levels in Expi293 cells (Thermo Fisher Scientific, Waltham, MA, USA).

Cloning into Vectors for Transient Expression AB0089 heavy and light chains were synthesized using Thermo Fisher Scientific's GeneArt gene synthesis platform and cloned into the pcDNA3.4 vector. The vector backbone for cloning the AB0089 chain S was to first use the pcDNA3.4 vector (Thermo Fisher Scientific, Waltham, Mass., USA) to create KpnI and BamHI restriction sites for linearization and New It was prepared by cloning using NEBuilder HiFi Technology from England Biolabs (Ipswich, Mass.). A general protocol for cloning using NEBuilder HiFi Technology was followed. Briefly, a gBlock (double-stranded DNA fragment) expressing the gene of interest was cloned into the vector backbone and transformed using TG-1 bacterial cells using the Zyppy Plasmid Miniprep Kit (Zymo Research, Irvine, Calif.). ) was used to recover the DNA and the plasmid sequence was confirmed by Sanger sequencing (Genewiz, South Plainfield, NJ). Once the sequence was confirmed, maxi-preps were performed to isolate plasmid DNA using the Nucleo Bond Xtra Maxi EF kit (Macherey-Nagel, Bethlehem, Pa.).

Expression Transient expression in Expi293 cells. performed according to a protocol. Expi293 cells were generated for transient expression of the three polypeptides comprising AB0089: pETseq0854 (chain H), pETseq0424 (chain L) and pETseq1739 (chain S) using a plasmid ratio of 4:1:1. A high proportion of heterodimers was achieved. Briefly, DNA and PEIpro reagent (Polyplus Transfection; Illkirch, France) are first mixed separately in OptiMEM media (Life Technologies, Thermo Fisher Scientific, Waltham, MA), then mixed and added to Expi293 cells. DNA-PEI complexes were made by this (>95% feasibility; 2.5×10 ⁶ /mL VCD). After transfection, cells were cultured at 37° C. and 8% CO ₂ in a humidified shaker incubator. Cell supernatants were harvested after 5 days.

Transient Expression in ExpiCHO Cells Transient transfection of ExpiCHO cells was performed according to the general protocol outlined by the manufacturer (Life Technologies, Thermo Fisher Scientific, Waltham, Mass.). ExpiCHO cells were engineered for expression of the three polypeptides comprising AB0089: pETseq0854 (chain H), pETseq0424 (chain L) and pETseq1739 (chain S) using a plasmid ratio of 4:1:1. One DNA strand is transfected to achieve a high percentage of heterodimers. Briefly, DNA-Expifectamine complexes were made by first mixing DNA and Expifectamine reagent (Life Technologies) separately in OptiPro medium (Life Technologies), then mixing and adding to ExpiCHO cells. (>95% viability; 6×10 6 /mL VCD). After transfection, cells were cultured at 37° C. and 8% CO ₂ in a humidified shaker incubator. ExpiCHO feed and enhancers were added after 18-20 hours to boost protein expression according to the protocol. Cell supernatants were harvested after 9-11 days, depending on cell viability (>70%).

Harvesting To harvest AB0089, the cell culture supernatant was transferred to 500 mL or 1 L polycarbonate centrifuge bottles, spun at 7500 rpm for 35 minutes at 4° C., and then filtered using a 0.2 micron filter bottle. . The following materials/reagents (suppliers; catalog numbers) were used:
●Vi-CELL XR cell counter (Beckman Coulter)
- Vi-CELL 4mL sample cup (Beckman Coulter; 08229NT2)
●Vi-Cell reagent pack (Beckman Coulter; B94987)
● 1 L polycarbonate centrifuge bottle (Beckman Coulter; 366751)
● 500 mL polycarbonate centrifuge bottle (Beckman Coulter; 355649)
- Nalgene® Rapid-Flow™ filter unit and bottle top filter, PES membrane, sterile, Thermo Scientific, 1 L (VWR; 73520-986)
- Nalgene® Rapid-Flow™ filter unit and bottle top filter, PES membrane, sterile, Thermo Scientific, 500 mL (VWR; 73520-984)

Purification Protein A Capture Chromatography A 5.0 cm diameter SNAP column was packed to a bed height of 5.6 cm using Protein A MabSelect SuRe gel to give a column volume of approximately 110 ml. Protein A method steps and buffers are shown in Table 121. Elution fractions were neutralized to approximately pH 7.0 using 1.0 M Tris, pH 8.3 (5-10% of elution volume). The following materials/reagents (suppliers; catalog numbers) were used:
● SNAP column (Essential Life Solutions; S-50/125-PASS-OE-FP10)
- MabSelect SuRe Protein A Gel (GE Healthcare; 175-43-802)
- 5x protein A binding buffer (500 mM sodium phosphate, 750 mM sodium chloride pH 7.2) (Boston Bioproducts; C-6332)
- 1x protein A binding buffer (100 mM sodium phosphate, 150 mM sodium chloride, pH 7.2) (5x stock dilution)
● 100 mM glycine pH 3.0 (Boston Bioproducts; BB-95-100 mM)
- 1.0 M Tris, pH 8.3 (Boston Bioproducts; LT-783)
● 10.0N sodium hydroxide (VWR; BDH7247-4)
● 0.1N sodium hydroxide (10.0N diluted stock)

Cation Exchange Chromatography Cation exchange chromatography (CIEX) was performed using Poros XS resin packed in an XK-26 column body to a bed height of approximately 24.5 cm with a final column volume of 130 ml. The equilibration and wash buffer (Buffer A) was 50 mM sodium acetate pH 5.0 with 10 mM sodium chloride. Elution buffer (Buffer B) was 50 mM sodium acetate pH 5.0 with 1.0 M sodium chloride.

The eluate from the Protein A column was diluted at least 5-fold with Buffer A to adjust pH and conductivity. After washing the loaded column, the protein was eluted with steps of increasing sodium chloride concentration. The steps of the IEX chromatography method are shown in Table 122.

Buffer Exchange by G25 Chromatography and Diafiltration The IEX product was packed in an XK-26 column to a bed height of 30 cm (160 ml column volume) on Sephadex™ G25 Fine resin (GE Healthcare, P/N 17-0032- 01) was buffer exchanged into PBS pH 7.4 (ThermoFisher Scientific, P/N 10010023) by gel filtration chromatography. Alternatively, the product was buffer exchanged and concentrated by discontinuous diafiltration using Amicon Ultra 15 centrifugal filters (Millipore, P/N: UFC903024).

SDS-PAGE
NuPAGE 4-12% Bis-Tris gels were run in MES running buffer. For both non-reduced and reduced preparations, wells were loaded with 2-3 μg of sample. Reduced samples were treated with 20 mM DTT and heated at 70° C. for 10 minutes. The gel power setting was constant at 200V for 50 minutes. Protein bands were visualized by staining with Novex SimplyBlue SafeStain according to the manufacturer's instructions. Gels were scanned with an Azure Biosystems C600 imager. The following materials/reagents (suppliers; catalog numbers) were used:
●XCell SureLock Mini-Cell (Thermo Fisher; EI0001)
PowerEase™ 300W power supply (Thermo Fisher; PS0301)
● Isotemp Digital Heat Block (Fisher Scientific; 88-860-022)
- NuPAGE MES SDS running buffer (20X) (Thermo Fisher; NP0002)
●Simply Blue SafeStain (Thermo Fisher; LC6065)
- NuPAGE LDS sample buffer (4X) (Thermo Fisher; NP0007)
●Novex Sharp Prestained Protein Standard (Thermo Fisher; LC5800)
● 2-mercaptoethanol (MP Biomedical; 194705)
- Azure c600 imager (Azure Biosystems)

Analytical size exclusion chromatography (Superdex 200)
Analytical size exclusion chromatography was performed using a Superdex 200 Increase 10/300 column attached to an Agilent 1260 HPLC system. The mobile phase was PBS pH 7.4 (ThermoFisher Scientific, P/N 10010023). The flow rate was 0.5 ml/min and the run time was 45 minutes. Absorbance was measured at 214 nm and 280 nm. Peaks were integrated by Open Lab CDS data analysis software version 2.3 using the GC/LC area percent method. Peaks below the detection threshold were integrated by manually defining a baseline.

Intact Mass 10 μg of AB0089 was diluted to 0.25 μg/μL with 0.1% formic acid and 1 μg was injected onto a MabPac 2.1×100 mM column and coupled to a QExactive+ mass spectrometer with the Biopharma option. was analyzed using a Thermo Vanquish UHPLC. Spectra were acquired in high mass range mode at a resolution of 17,500. Data were analyzed using the Sliding Window ReSpect algorithm of the BioPharma Finder data analysis software package. The following materials/reagents (suppliers; catalog numbers) were used: 0.1% formic acid in water (Honeywell; LC452-2.5) and MAbPac RP 2.1×100 mM, 4 μm column (Thermo Scientific; 088647).

Endotoxin (endotoxin)
Endotoxin readings were performed on an Endosafe® nexgen MCS cartridge reader (Charles River Laboratories) and cartridge (Charles River Laboratories P/N PTS2001F). Samples were prepared for assay by dilution into either endotoxin-free water or sterile PBS pH 7.4 (ThermoFisher Scientific, P/N 10010023). An assay was considered valid if all assay performance parameters and controls were within the specifications.

Hydrophobic interaction chromatography (HIC)
An injection of AB0089 (5 μg) was prepared in a 5:4 ratio of high salt buffer (100 mM sodium phosphate, 1.8 M ammonium sulfate, pH 6.5) to sample. Samples were analyzed using an Agilent 1260 Infinity II HPLC equipped with a Sepax Proteomix HIC Butyl-NP5 5uM column held at 25°C. The gradient was run from 0% low salt buffer (100 mM sodium phosphate, pH 6.5) to 100% low salt buffer over 6.5 minutes at a flow rate of 1.0 ML/min. Chromatograms were monitored at 280 nm. The following materials/reagents (suppliers; catalog numbers) were used:
- Proteomix HIC Butyl-NP5 5uM column, 4.6x35mM (Sepax; 431NP5-4603)
● Sodium dihydrogen phosphate (Sigma Aldrich; RES20906-A702X)
● Disodium hydrogen phosphate (Fisher Bioreagents; BB332-500)
Ammonium sulfate, >99.0% (Sigma; A4418-500G)
- Sodium hydroxide, 10.0N (Avantor Materials; 5000-03)
● Hydrochloric acid, 10.0 N (Ricca Chemical Company; 3770-32)

Non-reduced CE-SDS
AB0089 was diluted to 0.5 mg/mL using 1X sample buffer to achieve a final sample volume of 50 μL. Internal standards and iodoacetamide were added to the samples, which were incubated in a heat block at 70°C for 10 minutes, followed by an ice bath for 5 minutes. Samples were transferred to 96-well plates, centrifuged and loaded onto the instrument. Individual samples were loaded into capillaries at 4600 volts for 40 seconds and separated in a Maurice apparatus (ProteinSimple, San Jose, Calif.) at 5750 volts for 35 minutes. The following materials/reagents (suppliers; catalog numbers) were used:
● 25x internal standard (ProteinSimple; 046-016)
●CE-SDS Plus Cartridge (ProteinSimple; 090-157)
● Iodoacetamide (Sigma-Aldrich; 16125-5G)
Maurice CE-SDS PLUS Application Kit Reagents (Bottom Running Buffer, Separation Matrix, Wash Solution, Conditioning Solution 1 & 2, 1X Sample Buffer) (Protein Simple 7-5; 04)
● Top electrophoresis buffer (ProteinSimple; 046-384)

Reduced CE-SDS
AB0089 was diluted to 0.5 mg/mL using 1X sample buffer to achieve a final sample volume of 50 μL. An internal standard and beta-mercaptoethanol (Bio-Rad, #161-0710) were added to the samples and incubated in a heat block at 70° C. for 10 minutes followed by an ice bath for 5 minutes. Samples were transferred to 96-well plates, centrifuged and loaded onto the instrument. Individual samples were loaded into capillaries at 4600 volts for 40 seconds and separated on a Maurice apparatus (ProteinSimple, San Jose, Calif.) at 5750 volts for 31.2 minutes.

Capillary isoelectric focusing (cIEF)
AB0089 was diluted to 1 mg/mL using MilliQ water and 15 μL of sample was added to 60 μL of master mix (water, methylcellulose, Pharmalyte 3-10, arginine, pI markers 4.05 and 9.99) Vortex and briefly centrifuge. 60 μL of sample was aspirated from the top of the solution, added to a 96-well plate and centrifuged prior to testing. The sample was separated in a Maurice apparatus (ProteinSimple, San Jose, Calif.) at 1500 volts for 1 minute followed by 3000 volts for 8 minutes. The following materials/reagents (suppliers; catalog numbers) were used:
● 0.5% methyl cellulose (ProteinSimple; 102730)
● 1% methyl cellulose (ProteinSimple; 101876)
● 500 mM arginine (ProteinSimple; 042-691)
Anolyte (ProteinSimple; 102727)
Catholyte (ProteinSimple; 102728)
● cIEF cartridge (ProteinSimple; 090-101)
● Fluorescence calibration standard (ProteinSimple; 046-025)
● Pharmalyte 3-10 (Sigma Aldrich; P1522-25ML)
● pI marker 4.05 (ProteinSimple; 046-029)
● pI marker 9.99 (ProteinSimple; 046-034)
● System suitability mix (ProteinSimple; 046-027)
● System suitability peptide panel (ProteinSimple; 046-026)

Differential scanning calorimetry (DSC)
AB0089 was prepared at 0.5 mg/mL in 1X PBS (Gibco, #10010-031) or alternate formulations. 325 μL was added to a 96-well deep well plate along with matching buffer blanks. Thermograms were generated using a MicroCal PEAQ DSC (Malvern, Pa.). The temperature was increased from 20 to 100°C at 60°C/hour. Raw thermograms were background subtracted, a baseline model was splined, and a non-two state model was used to fit the data.

SPR instrument and reagents Biacore 8K (Instrument ID: 2222251, GE Healthcare Life Sciences) was used to perform reaction kinetic measurements and Biacore 8K Insight Evaluation Software was used to analyze experimental data. The following materials/reagents (suppliers; catalog numbers) were used:
- 10 mM sodium acetate, pH 5.0 (Cytiva; BR100351)
● 10 mM glycine, pH 1.7 (Boston BioProducts; BB-2413D)
● 10xHBS-EP+ (Cytiva; BR1006-69)
● 1xHBS-EP+ Prepared in-house from 10x stock solution ● 1xMBS-EP+, pH 6.0 (manufactured in-house)
- BSA Fraction V (7.5%) (Gibco; 15260-037)
● NaCl (Fisher Scientific; S271-1)
● NaOH (Cytiva; BR100358)
● Amine coupling kit (Cytiva; BR-1000-50)
● Mouse antibody capture kit (Cytiva; BR100838)
● CM5 chip (Cytiva; 29-1496-03)
● Series S sensor chip SA (Cytiva; BR-1005-31)
● SA chip (Cytiva; BR100531)
- Biotin capture kit (Cytiva; 28-9202-34)
- Goat anti-human IgG Fc cross-absorbing secondary antibody (Invitrogen; 31125)
● Micro Bio-Spin 30 column (Bio-Rad; 732-6223)
-Amicon Ultra 0.5mL centrifugal filter 10,000NMWL (Millipore Sigma; UFC501096)

CLEC12A Affinity by SPR Binding affinity of AB0089 to recombinant human CLEC12A (Dragonfly Tx) was measured by SPR using a Biacore 8K instrument. Briefly, human Fc-specific antibodies were covalently immobilized onto the carboxymethyldextran matrix of a CM5 biosensor chip via amine coupling chemistry at a density of approximately 8000-10000 resonance units (RU). , produced an anti-hFc IgG chip. AB0089 samples were captured onto an anti-hFc IgG chip at a concentration of 1.5 μg/mL at a flow rate of 10 μL/min for 60 seconds to achieve a capture level of approximately 150 RU. hCLEC12A-His was serially diluted (100 nM to 0.14 nM) in 3-fold SPR running buffer and injected over the captured test material at a flow rate of 30 μl/min. Association was monitored for 300 seconds and dissociation for 600 seconds. The surface was regenerated between cycles by injecting 3 pulses of 10 mM glycine-HCl, pH 1.7 at 100 μl/min for 20 seconds. HBS-EP+ (1X) containing 0.1 mg/ml BSA was used as the running buffer throughout the experiment. Experiments were performed at physiological temperature of 37°C. To obtain kinetic rate constants and overall binding affinities, the double-referenced data were fitted to a two-state kinetic model using Biacore 8K Insight Evaluation software (Cytiva). A justification for using the two-state model is shown below. The goodness of fit between the fitted curve and the experimental data was expressed by the evaluation software as ^chi2 .

NKG2D Affinity by SPR Binding affinity of AB0089 to recombinant human NKG2D (Dragonfly Tx) was measured by SPR using a Biacore 8K instrument. Briefly, mFc-specific antibodies were covalently immobilized via amine coupling chemistry onto the carboxymethyldextran matrix of CM5 biosensor chips at densities of approximately 8000-10000 RU to generate anti-mFc IgG chips. mFc-tagged human NKG2D was captured at a concentration of 0.2 μg/mL onto an anti-mFc IgG chip at a flow rate of 10 μL/min for 60 seconds to achieve a capture level of approximately 30 RU. AB0089 was buffer exchanged into HBS-EP+ (1X) buffer and serially diluted (5000 nM to 9.77 nM) in 2-fold dilutions with HBS-EP+. The analyte (AB0089) was injected over the captured test material at a flow rate of 30 μl/min. Association was monitored for 60 seconds and dissociation was monitored for 60 seconds. The surface was regenerated between cycles by injecting three pulses of 10 mM glycine-HCl, pH 1.7 at 100 μL/min for 20 seconds. HBS-EP+(1X) was used as the running buffer throughout the experiment. Experiments were performed at physiological temperature of 37°C. Double-referenced data are global fits with a 1:1 kinetic and steady-state interaction model and a 1:1 kinetic fit model using Biacore 8K Insight Evaluation software (GE Healthcare). Fitted by analysis. The steady-state fit offset was set to constant (0) because this sample had been buffer exchanged into running buffer prior to each experiment. Goodness of fit was expressed in the evaluation software as ^chi2 considered in the context of _Rmax values.

Synergistic effect of simultaneous binding of CD16a and NKG2D Synergistic binding of NKG2D and CD16a was assessed by SPR. hNKG2D alone (12.3 μg/mL), CD16a F158 allele alone (9 μg/mL), and a mixture of hNKG2D and CD16a F158 at the same concentration were each allowed to amine-couple to the surface of a CM5 Series S Biacore chip for 1 minute. rice field. 1.8 μM AB0089 or trastuzumab was infused at 10 μL/min for 150 seconds. The dissociation phase was observed for 180 seconds when regeneration was not required and for 1800 seconds when spontaneous regeneration of the surface (almost complete dissociation of the analyte) was required between cycles of the same flow rate. 1×HBS-EP+ buffer was used as running and sample dilution buffer.

Co-engagement (CLEC12A and NKG2D)
AB0089 was diluted in 1×HBS-EP+ buffer containing 0.1 mg/mL BSA and captured onto the anti-human Fc surface of a CM5 chip at a flow rate of 5 μL/min for 60 seconds to achieve a capture level of 150-250 RU. The net difference between the baseline signal and the signal after completion of AB0089 injection representing the amount of AB0089 captured was recorded. hCLEC12A-His (200 nM) or mFc-hNKG2D (7 μM) was injected into the captured AB0089 at 20 μl/min for 90 seconds to saturation. Immediately following this injection, a preincubated mixture of hCLEC12A-His (200 nM) and mFc-hNKG2D (7 μM) was injected at a flow rate of 20 μL/min for 90 seconds using the ABA injection command of the Biacore 8K control software. (the second target was pre-mixed with the first target to ensure that all binding sites of the first target were occupied). The chip was regenerated by two 20 second pulses of 10 mM glycine (pH 1.7) at 100 μL/min. This experiment was performed at 37° C. and 1×HBS-EP+ buffer containing 0.1 mg/ml BSA was used as running buffer. Binding of each antigen, expressed in RU, was recorded as the net difference between the baseline signal before and after injection of the individual antigen. A value of 1.0 was assigned to the average relative percent binding of each target bound to AB0089 that was not occupied by another target (injected first). The average relative binding stoichiometry of each target bound to captured AB0089 already saturated with other targets (second injection) is expressed as a percentage of the total capacity to bind unoccupied AB0089. was done.

Epitope binning Anti-CLEC12A antibodies or TriNKET were immobilized directly to the active flow cell of a series S CM5 sensor chip via amino coupling at a concentration of 3 μg/mL for final densities of 370-1070 RU. A classic sandwich assay performed at 25° C. was used for epitope binning. The first injection was a 120 second association of 200 nM CLEC12A-His followed directly by 200 nM sandwich antibody/TriNKET. The sandwich antibody/TriNKET had an association time of 120 seconds and a dissociation time of 120 seconds. Injections of both analyte and sandwich molecules were performed at 30 μL/min. A 20 second pulse of 10 mM glycine pH 1.7 injected at 100 μL/min was used to regenerate the chip surface after dissociation. The first cycle included buffer injections for both the analyte and the sandwich molecule. The second cycle included 200 nM hCLEC12A-His analyte followed by buffer injection for sandwich molecules. Subsequent cycles included injection of 200 nM hCLEC12A-His analyte followed by 200 nM sandwich molecules. 1×HBS-EP+running buffer was used throughout the experiment. Sensorgrams were visualized using Biacore Insight Evaluation software.

FcγR binding Human and cynomolgus monkey CD64, the high and low affinity alleles of CD16a (V158 and F158, respectively), two alleles of CD32a (H131 and R131), CD16b, CD32b and cynomolgus CD16 were primed Captured via biotin on a Biacore chip (according to manufacturer's instructions). Table 123 summarizes the capture level and type for each of the Biacore chips used. Since the interaction of streptavidin and biotin is irreversible, the capture step for all receptors except FcγRI (CD64) was performed only once before starting binding experiments. Capture of hCD64 and cyno CD64 on the CAP chip surface was performed during each cycle according to the Biotin CAPture kit manufacturer's instructions.

Prior to FcγR binding experiments requiring high starting concentrations, AB0089 and trastuzumab, samples were buffer exchanged into 1×HBS-EP+SPR running buffer by 3 serial dilution/concentration cycles using a 0.5 mL concentration unit. , diluted the original buffer components over 300-fold while maintaining high protein concentrations. Protein concentrations were measured at A280 on a NanoDrop instrument using the appropriate theoretical molar extinction coefficients of the samples (196790 M ⁻¹ cm ⁻¹ for AB0089 and standard IgG settings for trastuzumab).

Each FCγR binding experiment consisted of multiple cycles utilizing 2-fold serial dilutions of AB0089 or trastuzumab. A blank cycle of 0 nM and a chip surface without receptor placed at the start of each concentration series were used as references. Each experimental cycle included association, dissociation, and regeneration steps. Analyte (AB0089 or trastuzumab) concentrations, association and dissociation times, and regeneration parameters were FcγR specific (Table 123). All steps were performed at a flow rate of 30 μL/min and 25°C.

To stabilize the chip surface, a series of 2-5 blank experimental cycles were performed prior to sample analysis, substituting the appropriate running buffer for the analyte. Raw data were fitted to either a 1:1 kinetic model or a steady-state affinity model using Biacore Insight evaluation software. Goodness of fit was assessed using either the calculated _Rmax or ^chi2 (calculated by the software), which is associated with the apparent maximal binding response. All reported values were rounded to one decimal place after calculation.

FcRn Binding Recombinant human or cynomolgus monkey (cyno) FcRn was captured on the surface of SA series S chip for 1 minute at a concentration of 0.25 μg/mL. AB0089 and commercial trastuzumab (IgG1 assay control) were buffer exchanged to MBS-EP+, pH 6.0 prior to SPR binding experiments. Each FcRn binding experiment consisted of multiple cycles utilizing varying concentrations of the analyte. Two-fold dilution series of AB0089 and trastuzumab (12 μM-0.023 μM) were used. A blank cycle of 0 nM placed at the beginning of each concentration series and a blank amine coupling modified chip surface lacking FcRn were used as references. Each experimental cycle included association, dissociation, and regeneration steps. This association step consisted of a 60 second analyte injection followed by a 60 second dissociation step. Surface regeneration (60 sec injection of HBS-EP+buffer, pH 7.4) completed each kinetic cycle. All steps were performed at a flow rate of 30 μL/min and 25°C. MBS-EP+ buffer, pH 6.0 was used as running buffer. This data was fitted to a steady-state affinity model using the Biacore Insight evaluation software. The ^chi2 (calculated by the software) associated with the calculated R _max was used to assess the goodness of fit. All reported values are rounded to one decimal place.
KHYG-1-CD16aV-mediated cytotoxicity assay

The efficacy of AB0089 was tested on KHYG-1-CD16aV cells. EC50 and maximal lysis values were derived from cell lysis curves using GraphPad Prism software (4 parameter logistic nonlinear regression curve fitting model).

Cytotoxicity Assay Via Primary NK Cells The efficacy of AB0089 was tested on primary NK cells. EC50 values and maximal lysis values were derived from cell lysis curves using GraphPad Prism software (4 parameter logistic nonlinear regression curve fitting model).
Non-specific binding to polyspecific reagents (PSR)

50 μL of 100 nM TriNKET or control mAb in PBSF were incubated with 5 μL of pre-washed Protein A Dynabeads slurry for 30 minutes at room temperature. Magnetic beads bound to TriNKET or mAb were left on the magnetic rack for 60 seconds and the supernatant was discarded. Bound beads were washed with 100 μL PBSF. Beads were incubated with 50 μL of biotinylated PSR reagent diluted 25-fold from stock for 20 min on ice (Xu et. al., (2013) Protein engineering design and selection, 26, 663-670). Samples were placed on a magnetic rack, supernatant discarded and washed with 100 μL PBSF. Secondary FACS reagents for detecting binding of biotinylated PSR reagents to TriNKET or control mAbs were made as follows: 1:250 μL streptavidin-PE and 1:100 donkey anti-human Fc. , in PBSF. 100 μL of secondary reagent was added to each sample and allowed to incubate on ice for 20 minutes. Beads were washed twice with 100 μL PBSF. Samples were analyzed on a BDFACS Celesta. Two PSR controls, rituximab (PSR positive) and trastuzumab (PSR negative) were used in the assay. The following materials/reagents (suppliers; catalog numbers) were used: PBSF (1X PBS pH 7.4 + 0.25% BSA) (Dragonfly Tx), Dynabeads (Invitrogen; 10001D), Streptavidin-PE (Biolegend; 405204 ), and a donkey anti-human Fc Ab (Jackson ImmunoResearch; 709-096-149).
High-Spec® Cross-Reactivity Assay Using HuProt™ Arrays

The specificity of AB0089 was tested at concentrations of 0.1 μg/ml and 1 μg/ml against native HuProt human proteome arrays immobilized on microslides. Assays were performed at CDI Laboratories (Baltimore, Md.).

Immunogenicity Evaluation Cohen et al. (2013) Mol Cancer Ther, 12, 2748-59, EpiVax's EpiMatrix program was used for all calculations.

Molecular modeling Molecular modeling was performed using the SAbPred website (opig.stats.ox.ac.uk/webapps/newsabdab/sabpred/). CLEC12A and NKG2D binding arms were compared to 377 post-Phase I biotherapeutic molecules using Therapeutic Antibody Profiler (TAP). TAP used ABodyBuilder to generate a model of AB0089 with side chains from PEARS. CDRH3 was constructed by MODELER because this loop is the most diverse of all CDRs.

Five different parameters were evaluated:
1. Total length of CDR2. Patches of surface hydrophobicity (PSH) near CDRs3. Patches of positive charge (PPC) near the CDR4. Patches of negative charge (PNC) near the CDR5. Structural Fv charge symmetry parameters (sFvCSP) of CLEC12A-binding scFv and NKG2D-binding Fabs

Development Potential Accelerated (40° C.) in HST, pH 6.0 Stability AB0089 lot AB0089-002 was concentrated and treated with 20 mM histidine, 250 mM sucrose, 0.01% Tween using an Amicon Ultra 15 30K device -80, pH 6.0 (HST, pH 6.0). The concentration of collected samples was determined by A280 and samples were diluted to 20 mg/ml with HST buffer. A 0.2 ml aliquot was immediately frozen at -80°C as a control and two 0.15 ml aliquots were stored at 40°C in the dark. Aliquots were removed after 2 and 4 weeks and immediately frozen at -80°C until analysis. The following materials/reagents were used:
Amicon Ultra15 centrifugal filter (30K MWCO) (Millipore; UFC903024)
● Hydrochloric acid, 10N (RICCA; 3770-32)
● L-histidine (MP Biomedicals; 101954)
● Sodium hydroxide, 10N (JT Baker; 5000-03)
● Sucrose (VWR; M117-500G)
●Tween (registered trademark)-80 (VWR; 0442-1L)

Accelerated (40° C.) Stability in CST, pH 7.0 AB0089 lot AB0089-002 was concentrated and purified using an Amicon Ultra 15 30K device with 20 mM sodium citrate, 250 mM sucrose, 0.01% Tween®- 80, pH 7.0. The concentration of collected samples was determined by A280 and the samples were diluted to 20 mg/ml with CST buffer. A 0.4 ml aliquot was immediately frozen at -80°C as a control and four 0.15 ml aliquots were stored at 40°C in the dark. Aliquots were removed weekly and immediately frozen at −80° C. until analysis. The following materials/reagents were used:
Amicon Ultra15 centrifugal filter (30K MWCO) (Millipore; UFC903024)
● Hydrochloric acid, 10N (RICCA; 3770-32)
- Sodium citrate dihydrate (Fisher Chemical; S466)
● Sodium hydroxide, 10N (JT Baker; 5000-03)
● Sucrose (VWR; M117-500G)
●Tween (registered trademark)-80 solution (10%) (Sigma: PB192-10ML)

Forced Oxidation AB0089 lot AB0089-002 was diluted to 1 mg/ml using phosphate buffered saline (PBS). 6.7 μL of 3% hydrogen peroxide was added to a 1.0 ml aliquot of AB0089 to give a final concentration of 0.02% hydrogen peroxide. Oxidation of AB0089 was allowed to proceed for 24 hours at room temperature in the dark. After 24 hours, samples were buffer switched to PBS, pH 7.4 using an Amicon Ultra 0.5 mL 10K MWCO device according to the manufacturer's instructions. A non-oxidized control (0.5 mL) was stored at -80°C. The following materials/reagents were used: 30% hydrogen peroxide (VWR; BDH7690-1), Gibco PBS pH 7.4 (1x) (ThermoFisher; 10010-031), and Zeba desalting columns (Thermo Scientific; 89890).

pH 5 Stress Concentrated AB0089 lot AB0089-002 was diluted to 1.0 mg/ml using 20 mM sodium acetate, pH 5.0. Concentration was checked by A280 with a Nanodrop One spectrophotometer. Samples were split. Half was stored at -80°C as a control and the other half was incubated at 40°C for 2 weeks. After incubation, samples were stored at -80°C until analysis. Sodium acetate buffer (1 m, pH 5.0) (Boston Bioproducts; BB-60) was used.

pH 8 Stress Concentrate AB0089 lot AB0089-002 was diluted to 1.0 mg/ml using 20 mM Tris, pH 8.3. Concentration was checked by A280 with a Nanodrop One spectrophotometer. Samples were split. Half was stored at -80°C as a control and the other half was incubated at 40°C for 2 weeks. After incubation, samples were stored at -80°C until analysis. The pKa of Tris is temperature dependent. Tris (pH 8.3) measured at 25°C is expected to have a pH of about 8.0 at 40°C. Tris buffer (1 m, pH 8.3) (Boston Bioproducts; LT-783) was used.

Freeze/Thaw Three 0.1 ml aliquots of AB0089 lot AB0089-002 at 20 mg/ml in CST, pH 7.0 buffer were subjected to freeze/thaw cycles at −80° C. in Styrofoam containers to initiate the freezing process. slowed down. At cycles 2, 4, and 6, aliquots were removed and transferred to −80° C. storage until analysis.

Agitation 0.0 of 10 mg/ml AB0089 lot AB0089-002 in 20 mM sodium citrate, 250 mM sucrose, pH 7.0 or 20 mM sodium citrate, 250 mM sucrose, 0.01% Tween®-80, pH 7.0. A 5 ml aliquot was added to a deep well plate (Thermo, #278752), the place was sealed, covered with foil and shaken at 300 rpm at room temperature for 3 days. After 3 days the plates were spun down and analyzed by A280 (concentration), A340 (turbidity) and SEC (aggregation).

Low pH Retention AB0089 was captured on a MabSelectSure Protein A column and eluted with 100 mM glycine, pH 3.0. Peak fractions were pooled and 12 ml protein A eluate was adjusted to pH 3.3 with 100 mM glycine pH 3.0 buffer. The pH adjusted eluate was kept at room temperature. After 1.5 hours, the eluate was brought to neutral pH using 1.0 M Tris-HCl, pH 8.3. The pH-kept sample was further purified by cation exchange chromatography. The final product was buffer exchanged into PBS pH 7.4 for analysis and received designation AB0089 lot AB0089-003.

High Concentration AB0089 lot AB0089-002 was concentrated using a 30 kDa molecular weight cutoff device in PBS, pH 7.4. Concentration was determined by A280 on the Nanodrop and product quality was monitored by SEC as described above.

Size exclusion chromatography (TSKgel G3000SWxl)
The following methods were used to evaluate developability samples. 50 μg of test substance were injected into an Agilent 1260 Infinity II High Pressure Liquid Chromatography (HPLC) instrument equipped with a 1260 Quat Pump, 1260 Vialsampler, 1260 VWD. The sample was separated on a Tosoh TSKgel G3000SWxl, 7.8 mm x 30 cm ID, 5 μm column. SEC running buffer was PBS, pH 7.0, running at 0.50 ml/min. Absorbance was monitored at both 214 nm and 280 nm and peak areas were manually integrated and reported as percent high molecular weight species (HMWS), low molecular weight species (LMWS), and monomer. The following materials/reagents (supplier; catalog number) were used: gel filtration standards (Bio-Rad; 1511901), SEC buffer (20 mM sodium phosphate monobasic monohydrate/sodium phosphate dibasic heptahydrate) hydrate, 0.3m sodium chloride, pH 7.0) (Boston BioProducts; C-5194D), and TSKgel G3000SWxl, 7.8mm x 30cm, 5μm column (Tosoh; 08541).

LC-MS/MS Tryptic Peptide Mapping Briefly, 50 μg of AB0089 was diluted with 6 M guanidine hydrochloride in 250 mM Tris, pH 8.5, and disulfide bonds were removed with 25 mM dithiothreitol at 40° C. in a thermomixer. for 30 minutes. The reduced cysteines were then alkylated with 50 mM iodoacetamide for 60 minutes at room temperature in the dark. Reduced and alkylated proteins were buffer switched to 50 mM Tris, pH 8.0 and digested using trypsin (1:25 enzyme:substrate) at 37° C. for 1 hour. LC-MS/MS was run on a Thermo Vanquish UHPLC coupled to a QExactive+ mass spectrometer. Briefly, 5 μg of protein digest was separated on a Waters UPLC BEH Peptide C18 column (2.1×150 mM) over 150 min using a 2%-40% acetonitrile gradient at 0.3 ml/min. Mass spectra were acquired from 230-2000 m/z in positive mode at a resolution of 35,000 for full MS scans and 17,500 for Top 10 MS/MS scans. UPLC-MS/MS files were analyzed using the Byos PTM workflow (Protein Metrics). Peptides were identified using the correct mass and MS/MS searching for the AB0089 sequence. The peptide identification settings were as follows.
● Precursor mass error range 6.00 ppm
● Fragment mass error range 20.00 ppm
● Cleavage site(s) RK, C-terminal ● Digestion specificity Completely specific ● Missed cleavage 2
● modified carbamidomethyl/+57.021464 @ C, corrected ● DTT/+151.994915 @ C
● Oxidation/15.994915 @ M, W
● Dethiomethyl/-48.003371 @ M
● Deamidation/+0.984016 @ N, Q
● Pyro-Glu/-17.026549 @ NTerm Q
● Pyro-Glu/-18.010565 @ NTerm E
● Carbamyl/+43.005814 @ NTerm, K, R
●Acetyl/+42.010565 @ Protein NTerm
Ammonia loss/-17.026549 @N
●Dioxide/+31.989829@W
●Kynurenine/+3.994915@W
● N-glycan 50 common

The following materials/reagents (suppliers; catalog numbers) were used:
- 0.1% formic acid in acetonitrile (JT Baker; LC441-2.5)
- 0.1% formic acid in water (JT Baker; LC452-2.5)
- Formic acid (Thermo Scientific; 28905)
- Guanidine HCl (Alfa Aesar; A13543)
- Hydrochloric acid, 6 N (Avantor; 4103-01)
- Unweighed dithiothreitol (Thermo Scientific; 20291)
- Unweighed iodoacetamide (Thermo Scientific; 90034)
● Sodium hydroxide, 6N (Avantor; 5672-02)
● Tris Base (Avantor; 4109-01)
● Trypsin (Thermo Scientific; 90057)
- UPLC BEH Peptide C18 column (2.1 x 150 mM) (Waters; 186003556)
- Zeba spin desalting column (Thermo Scientific; 89882)

Results Discovery Hybridoma subclone 16B8. C8 (derived from sequence optimized AB0089 and its precursor AB0237) was developed through immunization of BALB/c mice with recombinant human CLEC12A-His protein (Figure 104). A detailed description of the immunization process, antibody screening, assessment of recombinant and cell surface expressed CLEC12A binding, and epitope binning is provided.

During the feasibility evaluation of AB0237, the liability for aspartate isomerization at CDRH3 of CLEC12A-binding scFv was confirmed. Attempts to remove this liability by single amino acid substitutions or by targeted yeast display approaches (randomization of 'DS' or 'S') resulted in low expression yields and significant loss of binding to CLEC12A, thereby , suggesting that this motif may be important for maintaining CDR structure. Therefore, to further improve the affinity of CLEC12A, affinity maturation efforts focused on optimizing CDRH3 and CDRH2, replacing DS sequences with DT in an optimized CDRH3 background. In addition, we sought to improve the human-likeness of the molecule by reducing the number of mouse backmutations within the framework. We hypothesized that only one backmutation at position H83 was necessary, whereas no other backmutations were essential to maintain the CDR architecture. An optimized scFv framework containing a single backmutation was named pET1596 and used as the background for all additional genetic manipulations. Subsequently, the (H94)K backmutation was restored, further enhancing the stabilization of the molecule. These engineering efforts have resulted in the discovery of AB0089 molecules that remove isomerization liability and improve humanness while retaining high affinity for human CLEC12A and potency against AML cancer cell lines.

（配列番号３２２）と比較して２つのアミノ酸の相違を含む１つのクローンは、親クローンよりもＣＬＥＣ１２Ａに対して高い結合親和性を示した（図１０５ Ｄ、Ｅ）。 Sequence Optimization A CDRH3-focused randomized affinity maturation library yeast display affinity maturation library (termed library 17) was generated at each position, one at a time, as described above. It was successfully generated by mutating the CDRH3 residue (YDYDDSLDY, SEQ ID NO: 139) of pET1596 or by changing two amino acid residues to all 20 amino acids. To enrich for scFv with higher affinity towards hCLEC12A in library 17, three rounds of selection were performed with biotinylated CLEC12A-His at 1 nM (first and second round of selection, Figure 105 A,B ) and at 0.1 nM (third round of selection; FIG. 105C). It was possible to compare the affinities between the parental clone pET1596 and the library clones and to accurately distinguish between them, allowing quantitative selection in real time (Fig. 105B,C,D). After three rounds of FACS sorting to isolate clones, parental clones

One clone containing two amino acid differences compared to (SEQ ID NO: 322) showed higher binding affinity for CLEC12A than the parental clone (Fig. 105 D,E).

（配列番号３２３），

（配列番号３２４）、

（配列番号３２５）、

（配列番号３２６）、

（配列番号３２７）、

（配列番号３２８）、

（配列番号３２９）、

（配列番号３３０）、

（配列番号３３１）、

（配列番号３３２）、

（配列番号３３３）、

（配列番号３３４）（ここでＸ＝任意のアミノ酸）に、最適化されたＣＤＲＨ３骨格中に組み込むことによって作成された。目標は、親クローン（ｐＥＴ１５９６）またはＣＤＲＨ３最適化バリアントよりも親和性の優れたバインダーを設計及び選択することであった。これにより、固定ＣＤＲＨ３を保持しながら、無作為化されたＣＤＲＨ２を使用してライブラリーを作成した。この親和性成熟酵母ディスプレイライブラリー（ライブラリー３０）で２ラウンドのＦＡＣＳソーティングを実施して、高親和性バインダーを濃縮した（図１０６）。 CDRH3-Focused Combinatorial Affinity Maturation Libraries Although results from CDRH3-focused libraries showed improved affinities, further improvements were highly desirable. Library 30 thus comprises a CDRH2 combinatorial library (WSGGK (SEQ ID NO: 138)

(SEQ ID NO: 323),

(SEQ ID NO:324),

(SEQ ID NO:325),

(SEQ ID NO:326),

(SEQ ID NO:327),

(SEQ ID NO:328),

(SEQ ID NO:329),

(SEQ ID NO: 330),

(SEQ ID NO:331),

(SEQ ID NO: 332),

(SEQ ID NO:333),

(SEQ ID NO:334) (where X=any amino acid) into an optimized CDRH3 scaffold. The goal was to design and select binders with better affinity than the parental clone (pET1596) or CDRH3 optimized variants. This generated a library using randomized CDRH2 while retaining fixed CDRH3. Two rounds of FACS sorting were performed on this affinity matured yeast display library (library 30) to enrich for high affinity binders (Figure 106).

（配列番号３２２）ＣＤＲＨ３バックグラウンド上のＣＤＲＨ２におけるＷＳＧＧＫ（配列番号１３８）から

（配列番号２７２）への変化を有する１つのクローンは、ＣＬＥＣ１２Ａに対して最も高い親和性を示した（図１０７）。このクローンは、親のｐＥＴ１５９６及びＡＢ０２３７と比較してＣＬＥＣ１２Ａ親和性の有意な改善を示した。 After two rounds of FACS sorting,

(SEQ ID NO:322) from WSGGK (SEQ ID NO:138) in CDRH2 on a CDRH3 background

One clone with a change to (SEQ ID NO:272) showed the highest affinity for CLEC12A (Figure 107). This clone showed significantly improved CLEC12A affinity compared to the parental pET1596 and AB0237.

（配列番号３２２）から

（配列番号２７３））を生成して、その結果、標的への高親和性結合を維持しながら、異性化のライアビリティを含まないＣＬＥＣ１２Ａ標的化ｓｃＦｖを得る。最適化されたＡＢ００８９分子とＡＢ０２３７のＣＤＲの比較を 表１２４に示す。

Restoration of DS Sequence Liability Once affinity matured clones were identified, site-directed mutagenesis was used to generate rational variants (CDRH3:

from (SEQ ID NO: 322)

(SEQ ID NO:273)), resulting in a CLEC12A-targeted scFv free of isomerization liability while maintaining high affinity binding to the target. A comparison of the CDRs of the optimized AB0089 molecule and AB0237 is shown in Table 124.

Sequence differences resulting from affinity maturation are underlined. Corrections for isomerization liability obtained by site-directed mutagenesis (DS to DT) are shown in green.

Based on structural modeling, it was decided to reintroduce a murine residue at position H94 to provide additional support for the CDR structure. AB0089 contains only two murine residues at positions H83 and H94 of the CLEC12A-binding scFv VH, whereas AB0237 has five murine backmutations. The remaining backmutations were returned to human germline, which improved the humanness of AB0089 (Table 125).

Molecular format and design AB0089 is a heterodimeric trifunctional antibody that redirects NK and T cell activity against cancer cells expressing CLEC12A. AB0089 contains three chains: chain H, chain L, and chain S. A schematic diagram of the AB0089 molecule is shown in FIG. Chain H is the heavy chain of the A49M-INKG2D targeting antibody that was chosen for its low affinity while being a potent agonist of the NKG2D receptor on NK cells and cytotoxic T cells. The M-I mutation was introduced to eliminate the Met102 residue of CDRH3, which is sensitive to oxidation and thus may present a potential liability. Chain L is the light chain of antibody A49M-I, which targets NKG2D. Chain S contains an scFv that binds with high affinity to CLEC12A on AML cancer cells. Both the scFv and Fab domains are fused to human IgG1 Fc. Both chains have several mutations in the CH3 domain introduced to ensure stable heterodimerization of the two Fc monomers. There are 7 such mutations in the IgG1Fc of AB0089: 3 mutations in chain H and 4 mutations in chain S. Two of these mutations are involved in the formation of stabilizing SS bridges (Y349C and S354C) between chains H and S (EU nomenclature). The scFv of chain S (VH-VL orientation) is stabilized by two mutations that introduce an SS bridge ((H44)C and (L100)C, Chothia nomenclature). Chain S also contains a (G ₄ S) ₄ non-immunogenic linker between VH and VL of the scFv. The amino acid sequence of the scFv targeting CLEC12A was humanized 16B8. Based on the sequence of the C8 antibody. The amino acid sequence of NKG2D Fab is based on the fully human antibody A49M-I obtained using human yeast display technology (Adimab).

Amino Acid Sequence The amino acid sequences of the three polypeptide chains that make up AB0089 are shown below. CDRs are defined using Chothia's nomenclature and are shown in Table 126. The C-terminal K, which is commonly processed and cleaved in IgG1, is deleted in chain S and chain H to prevent heterogeneity.

Chain S: CLEC12A-targeting scFv-CH2-CH3 (humanized sequence, CDRs in italics, backmutations in double underline, engineered scFv disulfides in lower case, and heterodimerization and engineered CH3 disulfides Fc mutations are single underlined).

（配列番号２７６）

(SEQ ID NO: 276)

Chain H: NKG2D targeting VH-CH1-CH2-CH3 (fully human, CDRs in italics, Fc mutations for heterodimerization, and engineered CH3 disulfides underlined).

（配列番号２６０）

(SEQ ID NO: 260)

Chain L: NKG2D-targeted VL-CL (fully human, CDRs shown in italics).

（配列番号２６１）

(SEQ ID NO: 261)

Analysis of potential sequence liability of CDRs Chain L (NKG2D-binding light chain) has no predicted liability. Chain H (NKG2D-bound heavy chain) has a DP in CDRH3. This motif is not subject to forced degradation (as with other constructs, data not shown). Therefore, this sequence is unchanged in AB0089. Chain S (CLEC12A binding scFv-Fc chain) has no predicted liability.

Description of heterodimerization mutations The EU numbering convention was used to annotate the amino acid residues of Fc.
Chain H contains the following mutations:
Heterodimerization mutations in the CH3 domain of the K360E-Fc region.
Heterodimerization mutations in the CH3 domain of the K409W-Fc region.
Y349C Intersubunit disulfide stabilizing mutation in the CH3 domain of the -Fc region.

The K360E/K409W mutations in the Fc region of chain H are designed to favor heterodimer interactions with chain S due to long range electrostatic interactions in addition to hydrophobic/conformational complementarity. (Choi et al., (2013) Mol Cancer Ther, 12, 2748-59; Choi et al., (2015) Mol Immunol, 65, 377-83). Mutation Y349C was introduced to generate an additional interchain disulfide bond Y349C-(CH3-chain H)-S354C(CH3-chain S) pair (Choi et al., (2015) Mol Immunol, 65, 377 -83).
Chain S contains the following mutations:
Disulfide-stabilizing mutations in the _VH region of G44C-scFv.
Disulfide-stabilizing mutations in the V _L region of S100C-scFv.
Heterodimerization mutations in the CH3 domain of the Q347R-Fc region.
D399V -Heterodimerization mutations in the CH3 domain of the Fc region.
A heterodimerization mutation in the CH3 domain of the F405T-Fc region.
S354C - An intersubunit disulfide stabilizing mutation in the CH3 domain of the Fc region.

Mutations G44C and S100C were introduced to stabilize the scFv by an engineered disulfide bond between VH and VL. Disulfide stabilization improves scFv structural and thermal stability. The Q347R/D399V/F405T mutations in the Fc region of chain S provide heterodimer-favorable interactions with chain H due to long-range electrostatic interactions in addition to hydrophobic/conformational complementarity (Choi et al, (2013) Mol Cancer Ther, 12, 2748-59). Mutation S354C was introduced to create an additional interchain CH3 disulfide bond with the Y349C (CH3-chain H)-S354C (CH3-chain S) pair (Choi et al, (2015) Mol Immunol, 65, 377 -83).

Heterodimerization of chain H and chain S occurs in the interacting pairs K409W (CH3A)-D399V/F405T (CH3B) (referred to as the W-VT pair) and K360E (CH3A)-Q347R (CH3B) (referred to as the ER pair). It is based on 5 mutations, the so-called EW-RVT, which have an asymmetrical hydrophobic interaction, which replaces the conserved electrostatic interaction with a It was designed to add asymmetric long-range electrostatic interactions (Choi et al., (2013) Mol Cancer Ther, 12, 2748-59). Detailed analysis of the CH3A-CH3B interface of the heterodimer revealed few structural perturbations due to mutations at the interface compared to native IgG1 Fc (FIG. 109). The backbone and side chain conformations of the residues flanking the interface mutations of Fc-EW-RVT are nearly identical to those of Fc-WT, except for the side chain substitutions of the mutated residues ( Figure 109A). The residues of the W-VT mutation pair, namely K409W(CH3A)-D399V(CH3B)/F405T(CH3B), are in close proximity to form complementary contacts at the mostly buried CH3A-CH3B interface (Fig. 109B). ), which suggests that the W-VT pair strengthens the hydrophobic interface between CH3A-CH3B by forming optimal hydrophobic interactions. Residues K360ECH3A-Q347RCH3B of the ER mutant pair are positioned close enough to 3.45 Å to form a salt bridge (Fig. 109C), unlike the wild-type KQ pair (4.61 Å), and the strong electrostatic Contributes to the preferential formation of heterodimers through interactions. Thus, the mutated residues introduced into Fc-EW-RVT form non-covalent interactions at the CH3A-CH3B interface, promoting heterodimer formation without major structural perturbations to the overall CH3 domain structure. .

By design, W-VT paired residues (K409W(CH3A)-D399V(CH3B)/F405T(CH3B)) and ER paired residues (K360E(CH3A)-Q347R(CH3B)) are placed at the heterodimeric CH3 interface. , forming asymmetric complementary hydrophobic and electrostatic interactions at 3.45 Å, respectively, which favor the heterodimer. On the other hand, the formation of each homodimer is due to steric clashes between 409WCH3A and F405CH3A residues and repulsive electrostatic interactions between 347R (CH3B) and K360 (CH3B) residues. It was thermodynamically unfavorable. Loss of the wild-type electrostatic interaction residues of K409WT-D39WT also inhibits homodimer formation. Further stabilization of the heterodimer is provided by the inter-CH3 disulfide bond EW-RVT (SS) variant with Y349C (CH3A) and S354C (CH3B) mutations (Figure 110). The X-ray structure of Fc-EW-RVT(S—S) has been depicted at 2.5 Å resolution (Choi et al., (2015) Mol Immunol, 65, 377-83) and is consistent with the known IgG1 structure. were very similar.

Structural Modeling In the absence of crystal structures, structural models of the variable segments of the CLEC12A and NKG2D binding arms were generated for comparison. Molecular modeling was performed using the SAbPred website (opig.stats.ox.ac.uk/webapps/newsabdab/sabpred/).

Surface charge distribution and hydrophobic patch analysis of CLEC12A binding arms of AB0089. panel). The charge distribution of the anti-CLEC12A scFv is polarized ('top view', bottom panel), with negatively charged residues predominantly within CDRH3 and CDRL2. The uneven distribution of electrostatic patches on the paratope is likely target-related and may reflect the complementarity of the charge distribution on the cognate epitope, which is similar to that of CLEC12A and AB0089. may contribute to the high-affinity interactions between scFvs.

Figure 112 shows analysis of the CDR length and surface hydrophobic patches of the CLEC12A targeting arm of AB0089. The analysis is based on the therapeutic antibody profile (TAP): opig. stats. ox. ac. uk/webapps/newsabdab/sabpred/tap. Candidates are compared with reference to 377 late stage therapeutic antibodies. The CDR length of the CLEC12A binding arm of AB0089 is within the normal range for late stage therapeutic antibodies. Hydrophobic patch analysis of CDRs of antibodies predicts their development potential. The theoretical hydrophobicity properties of the CLEC12A binding arm of AB0089 are well within the criteria for commercialized biotherapeutics and late-stage therapeutic antibody candidates.

FIG. 113 shows the analysis of patches containing positive and negative charges and the symmetry of these charges around the CDR regions. All three charts show that the charge distribution is within the norm compared to the therapeutic antibody population.

Surface charge distribution and hydrophobic patch analysis of the NKG2D binding arm of AB0089 The NKG2D binding Fab is shown in ribbon diagrams with three different orientations (Fig. 114, top panel) and the corresponding surface charge distributions at the same orientation are shown. (Fig. 114, bottom panel). The surface charge of the NKG2D binding arm is more evenly distributed than that of the CLEC12A targeting arm.

Figure 115 shows analysis of the CDR length and surface hydrophobic patches of the NKG2D targeting arm of AB0089. CDR length, hydrophobicity, and positive/negative charge distribution appear to be within the standards of existing therapeutic antibodies.

FIG. 116 shows the analysis of patches containing positive and negative charges and the symmetry of these charges around the CDR regions. All three charts show that this charge distribution is within the norm compared to the therapeutic antibody population.

Framework Evaluation The CLEC12A binding arm of AB0089 was discovered by immunizing mice. The frameworks chosen for CDR grafting are frequently used in clinical stage therapeutic monoclonal antibodies (Figure 117). The NKG2D binding arms are fully human, based on frameworks frequently used in advanced stage mAbs, and contain no aberrant residues or germline deviations.

Prediction of Immunogenicity The protein sequences of the three chains of AB0089 were examined for the presence of potentially immunogenic T-cell epitopes and ranked for immunogenic potential using the EpiMatrix algorithm (Figure 118). . AB0089 scores well on the Treg Modulated Immunogenicity Protein Scale. The Treg-adjusted EpiMatrix protein scores for the AB0089 three-chain sequences are −33.39 (chain H), −27.13 (chain S), and −23.49 (chain L) (Table 127). The predicted risk of immunogenicity of AB0089 appears to be low.

Expression Expression of AB0089 was achieved using the Dragonfly Tx F3'TriNKET platform purification method. Briefly, AB0089 was transformed into Expi293 and Expi293 by three plasmids encoding NKG2D-targeted light chain (chain L), NKG2D-targeted IgG heavy chain (chain H), and CLEC12A-targeted scFv-Fc fusion (chain S). It was expressed by transient transfection of ExpiCHO cells. For expression of both Expi293 and ExpiCHO, chain S:chain H:chain L were transfected at a plasmid ratio of 4:1:1 to maximize the proportion of the desired heterodimeric product. This ratio was obtained empirically based on previous humanized TriNKETs and was not specifically optimized for AB0089.

Purification Purification of AB0089 was performed using the F3'TriNKET platform purification method developed by Dragonfly Therapeutics (Figure 119). This purification process has been previously applied to material expressed in many different TriNKET Expi293, ExpiCHO, and CHO-M cell cultures. No optimization or extensive process development for AB0089 purification was performed. A two-step purification process was employed.

Two lots of AB0089 were produced in transiently transfected ExpiCHO and Expi293 cells. Table 128 summarizes the titer and final product purity of both lots. Purification of AB0089 lot AB0089-002 expressed in ExpiCHO is detailed below.

Protein A capture AB0089 was expressed in the ExpiCHO cell line as described above. Approximately 5.7 liters of filtered supernatant was loaded onto a protein A column. The load flow rate was 10 ml/min (298 cm/h; 3.3 min residence time). The yield of the capture step was approximately 752 mg. Figure 120 shows the chromatogram of the capture step.

According to SEC analysis, the protein A eluate contains predominantly the desired heterodimeric AB0089 species (approximately 88.8%) eluting at 23.4 minutes (Figure 121). Impurities include HMW aggregates and very small amounts of LMW.

SDS-PAGE analysis of protein A load, flow-through, and eluate indicates complete capture of AB0089 based on the absence of the AB0089 band in the flow-through sample. The Protein A elution lane shows mostly TriNKET, with additional bands corresponding to the HMW and LMW peaks observed by SEC (Figure 122).

Cation Exchange Chromatography The protein A eluate was purified by cation exchange chromatography using a Poros XS column (1.2 cmD x 20 cmH) operated at a flow rate of 7 ml/min (371 cm/h; 3.2 min residence time). further refined. When the conductivity reached about 20 mS/cm, the product started eluting with the 15% B elution step (Figure 123). SDS-PAGE analysis of the elution fractions shows high purity product in the first five fractions (Figure 124). The tail fraction of the peak was excluded from the IEX product pool as SDS-PAGE indicated the presence of contaminating product-associated proteins.

Concentration of the IEX fractions gave approximately 401 mg of purified AB0089. Pooled fractions were buffer exchanged into PBS pH 7.4 by dialysis. AB0089 yield after buffer exchange was approximately 391 mg at a final concentration of 9.66 mg/ml (AB0089 lot AB0089-002). Yields and recoveries for AB0089 lot AB0089-002 production are summarized in Table 129.

Characterization of AB0089 Lot AB0089-002 Used in Cyno Studies AB0089 Lot AB0089-002 intended for use in cyno studies was extensively characterized for purity, identity, binding to target, and potency. was taken. All standards tested met specifications.

Purity by SDS-PAGE SDS-PAGE of the final sample showed that the major band was consistent with the expected molecular weight for both reduced and non-reduced AB0089 (Figure 125).

Purity by analytical SEC (Superdex 200)
SEC analysis of purified AB0089 lot AB0089-002 showed 100% monomer as determined by integrated peak area (Figure 126).

Identity by Intact Mass Spectrometry The identity of AB0089 (lot AB0089-002) was confirmed by mass spectrometry (Figure 127). LC-MS analysis of intact AB0089 showed an observed mass (126,130.0 Da) in good agreement with a theoretical mass of 126,129.1 Da. The predominant glycosylation pattern observed (G0F/G0F) is typical for Fc-containing molecules expressed in CHO cells.

Endotoxin The final AB0089 lot AB0089-002 endotoxin level was 0.259 EU/mg. All assay parameters met specifications.

Biochemical and Biophysical Properties AB0089 is characterized by SEC, CE, cIEF, HIC, and DSC, with suitable purity and pre-development properties of an excellent biotherapeutic is backed by

Purity Purity of AB0089 was determined by CE-SDS (Figure 128). Under non-reducing conditions, the major impurities are induced by the method (free light chain and TriNKET-LC). Under reducing conditions, three expected chains (LC, HC and scFv-Fc chains) are observed. The purity of AB0089 Lot AB0089-002 as determined by SEC, NR, and RCE-SDS is summarized in Table 130.

Experimental pI and charge profile by cIEF The charge profile of AB0089 was analyzed by cIEF (Figure 129). AB0089 showed a major peak with a pI of 8.52±0.0. Some smaller, overlapping acidic and minor basic peaks are also observed.

Hydrophobicity assessed by HIC.
Hydrophobicity of AB0089 was assessed by HIC (Figure 130, Table 131). The chromatogram showed a single narrow peak, indicating that the molecule was very pure and homogeneous. The retention time of HIC was 10.2 minutes, which is within the normal behavior of commercially available monoclonal antibodies (8.9-13.5 minutes, data not shown).

Thermal stability by DSC Thermal stability of AB0089 was evaluated by DSC in several buffers (PBS pH 7.4, HST pH 6.0, CST pH 7.0). AB0089 showed high thermal stability in all buffers tested (Figure 131). The T _m 1 corresponding to scFv development met the criteria of >65° C. utilized for thermal stability assessment (Table 132).

Assignment of Disulfide Bonds AB0089 is an engineered molecule based on the backbone of a monoclonal IgG1 antibody. A typical IgG1 contains 16 disulfide bonds, whereas the F3' format of AB0089 has only 15 disulfide bonds. Half of AB0089 is an anti-NKG2D half antibody and thus contains the expected seven disulfide bonds (one in each IgG domain and an intermolecular disulfide connecting the LC to the HC). The other half of AB0089 is an anti-CLEC12A scFv-Fc fusion. The scFv contains three disulfide bonds (one in each anti-CLEC12A VH and VL domain and an engineered stabilizing disulfide that bridges the variable domains) and the Fc contains two predicted disulfides in the IgG domain. be The heterodimer is covalently linked with two native IgG1 hinge disulfides and one additional intermolecular engineered disulfide connecting Fab-Fc to scFv-Fc in the CH3 domain. Collectively, this results in 15 predicted disulfide bonds in AB0089. A predicted disulfide map of AB0089 is shown in FIG.

Disulfide bond connectivity of AB0089 was confirmed by LC-MS/MS peptide mapping analysis of non-reduced digests. Disulfide-linked peptides were identified by MS/MS database searching and comparing intensities in native and reduced digestion. In tryptic digestion, the VL and the engineered disulfide pair are connected by a long peptide containing two cysteines (three peptides connected by two disulfide bonds, S7:S36:S22), which is not observed in the LC-MSMS data. be. To detect engineered scFv-stabilizing disulfides, AB0089 was digested with both trypsin and chymotrypsin. Figure 133 shows extracted ion chromatograms (XIC) of engineered disulfide pairs of scFv (non-reduced and reduced) and the strongest charge states of the peptide pairs. The VL disulfide pair was not observed in the trypsin/chymotrypsin double digestion, probably too small and hydrophilic to be retained on the column. All other disulfides were identified after digestion with trypsin alone and contained engineered S—S bridges introduced to stabilize Fc heterodimerization (FIG. 134).

A summary of disulfide-linked peptides observed in AB0089 is shown in Table 133. All observed disulfide-linked peptides had high mass accuracy (<4 ppm), were reducible, and were sequence-verified by MS/MS fragmentation.

Binding Characteristics Binding Affinity to Human CLEC12A The affinity of AB0089 for human CLEC12A was determined by SPR at 37°C. AB0089 binds to human CLEC12A with high affinity (Figure 135 and Table 134). However, the binding exhibits heterogeneity and does not follow normal 1:1 binding kinetics. The experiments described below and in Figure 136 establish a two-state coupling mechanism and justify the use of a two-state model to fit the data. Similar two-state kinetics were observed with the CLEC12A binding arm of tepositamab (Merus).

The two-state binding model assumes two binding stages defined by two sets of rate constants.
1. Formation of the initial CLEC12A-AB0089 complex2. transition from initial complex to more stable final complex

This mechanism implies that the longer the association time in SPR experiments, the higher the proportion of more stable complexes at the surface and the slower the apparent dissociation rate. Both AB0089 and tepositamab-based molecules have association time-dependent apparent dissociation rates, thus supporting a two-state interaction model (Figure 136).

Binding to CLEC12A+ Isogenic and Cancer Cell Lines Evaluation of AB0089 binding to isogenic cell lines expressing CLEC12A and AML cancer cells was monitored by FACS (Figure 137, Table 135). AB0089 exhibits high affinity for cell surface expressed CLEC12A and binds to AML cell lines PL-21 and HL-60 with an EC50 of less than 1 nM. The specificity of AB0089 for CLEC12A was demonstrated by the lack of binding to parental Ba/F3 cells that do not express the target.

Binding affinity for polymorphic variants of human CLEC12A (Q244)
The polymorphic variant CLEC12A (Q244) is prevalent in 30% of the population. We tested the binding of AB0089 to this genetic variant by SPR and compared its affinity to the predominant form of CLEC12A (K244) (Figure 138). The binding affinities of AB0089 to the WT and Q244 variants of CLEC12A were similar (Table 136). Binding of AB0089 to cynomolgus CLEC12A (cCLEC12A) was also tested. No binding was observed at a concentration of 100 nM, indicating that AB0089 has no cross-reactivity with cynomolgus CLEC12A.

Binding to Differentially Glycosylated CLEC12A Human CLEC12A is a highly glycosylated protein (6 potential N-glycosylation sites with a missing extracellular domain (ECD) of 201 amino acids). Variation in the glycosylation status of CLEC12A on the surface of different cell types has been described in the literature (Marshall et al., (2006) Eur. J. Immunol., 36:2159-2169). CLEC12A expressed on the surface of AML cells from different patients may have different glycosylation patterns as well. To determine whether AB0089 binds to different glycovariants of human CLEC12A, the antigen was treated with neuraminidase (to remove terminal sialic acid) or PNGaseF (to remove N-linked glycans). Affinity was compared to untreated CLEC12A by SPR (Figure 139 and Table 137). AB0089 is expected to bind both fully glycosylated and deglycosylated CLEC12A, insensitive to heterogeneity in target glycan composition.

Binding Affinity to Human NKG2D AB0089 was tested for binding to human NKG2D ECD by SPR (Biacore) at 37° C. (FIG. 140). Recombinant mFc-tagged NKG2D dimers were used for this experiment, as NKG2D is dimeric in nature. The geometry of the binding sensorgrams shown in FIG. 140 enabled the calculation of affinity and kinetic data with two different fitting models: a steady-state affinity fit and a 1:1 kinetic fit. Rate constants and equilibrium affinity values are shown in Table 138. AB0089 was designed to bind human NKG2D with low affinity and, most importantly, with a fast dissociation rate. The dissociation rate constant was 1.43±0.03×10 ⁻¹ s ⁻¹ . Affinity values (K _D ) obtained by fitting the data to 1:1 kinetic and steady-state affinity models were very similar, 881 ± 10 nM and 884 ± 11 nM, respectively. , suggesting that the measured parameters are highly reliable.

Binding Affinity for CD16a (FcγRIIIa) One mode of AB0089 mechanism of action is through engagement of human CD16a (FCγRIIIa) expressed on NK cells via its Fc. Binding of AB0089 to CD16a (V158 allele) was assessed by SPR and compared to CD16a (V158) affinity for Trastuzumab, an approved and marketed monoclonal antibody therapeutic of the same IgG1 isotype (Figure 141, Table 139). Binding of AB0089 to CD16a is comparable to that of trastuzumab. Because AB0089 therapeutic efficacy relies on the synergistic effect of simultaneous engagement of two targets on NK cells, CD16 and NKG2D, a 3.8-fold difference in affinity is unlikely to have physiological consequences. , is also unlikely to affect AB0089 therapeutic efficacy. Additionally, slightly weaker binding to CD16a may also contribute to drug safety.

Binding to Fcγ Receptors As AB0089 is an IgG1-derived biologic drug candidate, it is important to maintain proper Fc receptor binding properties. SPR data suggest that AB0089 binds to human and cynomolgus monkey Fcγ receptors with affinity comparable to trastuzumab, a commercial IgG1 biologic that serves as an experimental control (Table 140). A detailed description of each individual receptor binding is provided below.

Binding of AB0089 to recombinant human and cynomolgus monkey CD64 (FCγRI)
Figures 142 and 143 show the binding of AB0089 to human and cynomolgus monkey CD64, respectively. Table 141 shows that AB0089 binds to recombinant human and cynomolgus monkey CD64 with affinities comparable to trastuzumab (less than 2-fold difference).

Binding of AB0089 to recombinant human CD32A (FCγRIIa)
Figures 144 and 145 show the binding of AB0089 and trastuzumab, respectively, to CD32a (H131 and R131 alleles) as tested by SPR. There is no meaningful difference between the allelic affinities of both AB0089 and trastuzumab (Table 142).

AB0089 Binding to Human CD16a (FCγR3a) and Cynomolgus Monkey CD16 (FCγR3) A detailed description of AB0089 binding to high affinity CD16a (V158) is provided herein. The binding of AB0089 to the F158 allele of human CD16a and cynomolgus monkey CD16 is shown in Table 143, Figures 146 and 147. AB0089 binding to recombinant human CD16a (V158 and F158 alleles) and cynomolgus monkey CD16 is comparable to the commercial IgG1 biologic drug trastuzumab. The 2.3-3.8 difference in affinity for the CD16 protein between AB0089 and trastuzumab is unlikely to have physiological consequences and is unlikely to affect therapeutic efficacy. This is because therapeutic efficacy relies on the synergistic effect of the engagement of two targets on NK cells (CD16a and NKG2D). The slight difference in apparent maximal binding response between trastuzumab and AB0089 is due to the difference in molecular weight. Furthermore, some replicates may show small variations in apparent maximal binding response caused by small variations in receptor capture levels. The shape of the sensorgram representing binding to the human CD16aF158 allele fits the interaction to both steady-state and 1:1 kinetic models, although the affinity-fit (steady-state) model is the historical Note that it was used for a reason.

Binding of AB0089 to human CD32B (FCγR2b)
Figure 148 shows binding of AB0089 and trastuzumab to CD32b as examined by SPR. There is no meaningful difference between the affinity of CD32b for AB0089 and trastuzumab (Table 144).

Binding of AB0089 to human CD16B (FCγR3b)
Figure 149 shows binding of AB0089 and trastuzumab to CD16b as examined by SPR. AB0089 binds human CD16b (FCγR3b) comparably to trastuzumab (Table 145). The 2.3-fold difference in affinity for the receptor is unlikely to be physiologically significant.

Binding to FcRn Figures 150 and 151 show binding of AB0089 to human and cynomolgus monkey (cyno) FcRn tested by SPR, respectively. The affinity of AB0089 for human and cynomolgus monkey FcRn is similar to that of the commercial IgG1 biologic, trastuzumab, which served as an experimental control (Table 146).

Simultaneous Engagement of AB0089 to CD16a and NKG2D AB0089 is a trifunctional IgG-based biologic with two modes of binding to NK cells. CD16a binding is accomplished via Fc and NKG2D binding via A49M-IFab. Both interactions are important for optimal cytotoxic activity of AB0089. To demonstrate the synergistic effect of co-engagement of human CD16a and human NKG2D binding, we performed SPR experiments to qualitatively compare AB0089 binding to NKG2D, CD16a, and mixed NKG2D-CD16a Biacore chip surfaces. rice field. Although the affinity of AB0089 for human NKG2D and human CD16a are both low, simultaneous binding to both targets results in an affinity effect manifested as a slower off-rate. Therefore, AB0089 may strongly engage CD16a and NKG2D (Fig. 152).

Co-engagement of CLEC12A and NKG2D. was continuously infused (Fig. 153). Target binding sensorgrams show that CLEC12A domain occupancy does not interfere with NKG2D binding (Figure 153, upper panel) and vice versa (Figure 153, lower panel). Kinetic parameters are not significantly affected by target occupancy of the AB0089 molecule due to the similarity in shape of the respective sensorgram segments showing binding of each target to free AB0089 and AB0089 saturated with other targets. is suggested. For example, the shape of the CLEC12A binding segment in the sensorgrams is similar in both panels. Note that a saturating concentration of NKG2D must be maintained throughout the experiment, shown in the bottom panel, due to the fast dissociation rate of the target. Furthermore, the lack of effect on the relative stoichiometry of each target binding (when compared to binding to unoccupied AB0089) suggests that the NKG2D and CLEC12A binding sites on AB0089 are completely independent. (Table 147). Therefore, AB0089 can successfully achieve simultaneous co-engagement of the tumor antigen and the NKG2D targeting arm.

Epitope binning We compared the binding epitopes of AB0237, AB0192 and AB0089 associated with three CLEC12A binding reference TriNKETs by SPR. cFAE-A49. Tepoditamab is based on the CLEC12A binding arm of the Merus molecule. (WO_2014_051433_A1), cFAE-A49. CLL1-Merus is based on Merus clone 4331 (WO_2014_051433_A1) and cFAE-A49. h6E7 is based on the h6E7 mAb from Genentech (h6E7). (U.S. Patent Application Publication No. 2016_0075787_A1). The interaction of AB0089 with hCLEC12A was blocked by AB0237 and two control molecules cFAE-A49. CLL1-Merus and cFAE-A49. Tepositamab is not a Genentech-h6E7-based molecule (Figure 154), suggesting that the binding epitope of AB0089 overlaps with that of tepositamab. Results indicated that both AB0089 and AB0237 share the same epitope. This footprint is cFAE-A49. Unlike AB0192, which further cross-blocks at h6E7.

Efficacy The efficacy of AB0089 was tested using the KHYG-1-CD16aV cytotoxicity assay using NK cell lines engineered to stably express CD16aV and NKG2D (Figure 155 and Table 148). AB0089 showed sub-nanomolar potency in promoting lysis of HL60 and PL-21 AML cells. Moreover, AB0089 potently enhances the cytotoxic lysis of PL21 AML cells mediated by purified human primary NK cells significantly over the corresponding monoclonal antibodies (Figure 156 and Table 149). Additional cytotoxicity evaluation of AB0089 is described in detail in Example 18.

Specificity Non-Specific Binding Assessed by PSR To assess the specificity of AB0089, a flow cytometry-based PSR assay measuring binding to preparations of detergent-solubilized CHO cell membrane proteins was performed. (Fig. 157). PSR assays have been demonstrated to correlate with orthogonal specificity assays such as cross-interaction chromatography, and baculovirus particle enzyme-linked immunosorbent assays. Behavior in these assays is strongly associated with antibody solubility and in vivo clearance, and therefore serves as a good predictor of developability (Xu et al., (2013) Protein engineering design and selection, 26, 663-670). AB0089 showed a very similar profile to the low PSR control (trastuzumab), suggesting high specificity and lack of non-specific binding to irrelevant proteins (Figure 157B). Rituximab was used as a positive control for PSR binding.

Specificity Evaluation of AB0089 on HuProt™ Arrays To directly examine the specificity of AB0089, protein array technology was explored. The HuProt™ Human Proteome Microarray provides the largest database of individually purified full-length human proteins on a single microscope slide. An array consisting of 22,000 full-length human proteins was generated from yeast S. cerevisiae. After being expressed and purified in cerevisiae, they are duplicate printed on microarray slides that allow profiling of thousands of interactions in a high-throughput manner. Figure 158 shows the relative binding (Z-score) of AB0089 at 1 μg/ml to human CLEC12A compared to the entire human proteome microarray. For comparison purposes, the top 24 proteins with remaining background binding to AB0089 were also provided. Table 150 shows the Z-score and S-score of AB0089 against human CLEC12A and the top 6 proteins on the microarray. The S-score is the difference between the Z-score of a given protein and one rank next to it. Based on Z and S score criteria, AB0089 showed high specificity for human CLEC12A and lack of off-target binding in the HuProt™ Human Proteome Assay.

Development feasibility evaluation The stability and forced degradation of AB0089 were evaluated under the following conditions.
Accelerated (40°C) Stability - 20 mg/ml AB0089 (20 mM histidine, 250 mM sucrose, 0.01% Tween®-80, pH 6.0) in formulation HST, pH 6.0, 40°C, 4 weeks (SEC, SPR, potency, CE-SDS, peptide map).
- 20 mg/ml AB0089 (20 mM sodium citrate, 250 mM sucrose, 0.01% Tween®-80, pH 7.0) in CST, pH 7.0, 40°C, 4 weeks (SEC, SPR, potency, CE-SDS, peptide map).
• Chemical stability - oxidation: 1 mg/ml AB0089 in PBS with 0.02% hydrogen peroxide, room temperature, 24 hours (SEC, CE-SDS, SPR, potency, peptide map).
- pH 8.0: 20 mM Tris, 40°C, 1 mg/ml AB0089 (cIEF, SPR, potency, peptide map) for 2 weeks.
- AB0089 at 1 mg/ml in pH 5.0:20 mM sodium acetate, 40°C, 2 weeks (cIEF, SPR, potency, peptide map).
Manufacturability - Freeze/Thaw: 20 mg/ml AB0089 in 20 mM sodium citrate, 250 mM sucrose, 0.01% Tween®-80, pH 7.0 (SEC, SDS-CE)
- Agitation: 10 mg/ml AB0089 in 20 mM sodium citrate, 250 mM sucrose, 0.01% Tween®-80, pH 7.0 (SEC, SDS-CE)
- High concentration: >150 mg/ml, PBS (SEC, A280)
- Low pH hold (mimicking the conditions of the viral clearance step of the manufacturing process): pH 3.3, 1.5 hours, ProA eluate (SEC); fully purified AB0089 (cIEF, CE-SDS, SPR, potency )
- Low pH retention (to investigate isomerization): fully purified AB0089 was diluted with ProA elution buffer and pH adjusted to 3.5. Samples were kept at room temperature for 1 hour, neutralized with Tris buffer and buffer exchanged to PBS, pH 7.4 before additional analysis (SEC, cIEF, CE-SDS, SPR, potency, peptide map ).

Accelerated (40° C.) Stability To assess the stability of AB0089, the molecule was treated with (1) 20 mM histidine, 250 mM sucrose, 0.01% PS-80, pH 6.0 (HST), and (2) 20 mM 4 weeks at 40° C. under two different buffer conditions of citrate, 250 mM sucrose, 0.01% PS-80, pH 7.0 (CST). Staged at a protein concentration of 20 mg/ml. Stability of both buffers was evaluated by SEC, CE, SPR and potency.

Stability in HST, pH 6.0 AB0089 showed high stability after incubation in HST, pH 6.0, 40°C for 4 weeks. No aggregation and minimal monomer loss (1.6%) were observed by SEC (Figure 159 and Table 151). Minimal fragmentation was detected by R CE-SDS (Figure 160 and Table 152). Furthermore, no significant differences in the amount of active species, binding kinetics, or affinity for hCLEC12A, hNKG2D, and hCD16a were observed between control and stress samples (Figure 161 and Table 153). Finally, no differences in potency between control and stressed samples were detected (Figure 162 and Table 154).

Stability at CST pH 7.0 AB0089 showed high stability after 4 weeks of incubation at CST, pH 7.0, 40°C. A minimal loss of monomer content (1.6%) was observed by SEC (Figure 163 and Table 155). No fragmentation of AB0089 was observed in CE-SDS (Figure 164 and Table 156). Furthermore, there were no significant differences in active protein content or binding affinities for hCLEC12A, hNKG2D, and hCD16a between control and stress samples (Figure 165 and Table 157). Finally, long-term heat stress did not affect the potency of AB0089 (Figure 166 and Table 158).

Peptide Map Analysis of Accelerated Stability Samples Control and 4-week stressed samples were analyzed by LC-MS/MS to determine whether 40° C. stress induced modification of complementarity determining regions (CDRs). Analyzed by peptide mapping. Based on peptide map data, all CDRs of AB0089 are resistant to modification upon high temperature stress (Table 159). Based on manual inspection of peak shapes, no evidence of aspartate isomerization was observed in peptides containing CLEC12A-bound CDRH3 (Figure 167).

Chemical Stability Forced Oxidation To assess the stability of AB0089 under oxidative stress, AB0089 was incubated with 0.02% hydrogen peroxide in PBS for 24 hours at room temperature. SEC analysis showed no difference in monomer content between oxidized AB0089 and control samples (Figure 168 and Table 160). No increase in fragmentation was detected with AB0089 by both reduced and non-reduced CE-SDS (Figure 169 and Table 161).

Site-specific oxidation of methionine was monitored by tryptic peptide mapping after forced oxidation. Significant levels of oxidation were detected only at the two methionines of Fc (Table 162). Our historical data show that the same residues are modified in 61.6% and 39.1% of trastuzumab under the same conditions.

Oxidation of tryptophan residues in AB0089 CDRs was also monitored in the same experiment. None of the tryptophan residues had significantly increased oxidation after stress (Table 163).

Oxidative stress did not significantly affect active protein content or affinity for hCLEC12A, hNKG2D, and hCD16aV (Figure 170 and Table 164). No significant effect of oxidation on AB0089 potency was observed (Figure 171 and Table 165).

pH 8 stress The chemical stability of AB0089 was assessed by long-term incubation at high pH (20 mM Tris, pH 8.0, 40° C., 2 weeks). AB0089 appears to be very stable: only 1.2% monomer loss was detected by SEC (Figure 172 and Table 166).

Reduced and non-reduced CE-SDS showed low levels of degradation of 1.7% and 3.9%, respectively (Figure 173 and Table 167).

After prolonged high pH stress, there was an acidic shift in the overall charge profile detected by cIEF (Figure 174 and Table 168). This acidic shift can be attributed to deamidation of the entire AB0089 sequence. A similar acidic shift was observed with trastuzumab after the same stress conditions (data not shown).

There were no significant differences in the amount or affinity of active protein for hCLEC12A, hNKG2D, or hCD16aV between stressed and control samples (Figure 175 and Table 169).

After prolonged exposure to high pH, the potency of AB0089 decreased approximately two-fold (Figure 176 and Table 170). Given that the orthogonal analysis methods showed no differences in binding or net species content (Figure 175), it is likely that these differences may be due to potency assay variability.

pH 5 Stress The chemical stability of AB0089 was also evaluated by long-term incubation at low pH (20 mM sodium acetate, pH 5.0, 40° C., 2 weeks). DF appeared to be very stable: only 0.3% loss of monomer was detected by SEC (Figure 177 and Table 171).

After prolonged pH 5 stress, low levels of AB0089 degradation were detected by non-reduced and reduced CE-SDS: 1.0% and 0.3% loss of monomer content, respectively (Figure 178 and Table 172).

After prolonged pH 5 stress, there was a small acidic shift in the overall charge profile detected by cIEF (Figure 178 and Table 172), with a significant increase in one acidic variant in particular.

There was no significant difference in active protein content or AB0089 affinity to hCLEC12A, hNKG2D, or hCD16aV after prolonged low pH exposure (Figure 180 and Table 174).

The efficacy of AB0089 after prolonged pH 5 exposure remained similar to that of control samples (Figure 181 and Table 175).

Peptide Mapping Analysis of pH 5 and pH 8 Stressed Substances To determine whether the pH stress-induced changes observed by cIEF are due to complementarity determining region (CDR) modifications (alterations), pH 5 and pH 8 stress samples were analyzed by LC-MS/MS peptide map. Based on peptide map data, all CDRs of AB0089 are resistant to modification during low and high pH stress (Table 176). Based on manual inspection of peak shapes, no evidence of aspartate isomerization was observed in peptides containing CDRH3 of the CLEC12A targeting arm of AB0089 (Figure 182).

Manufacturability Freeze/Thaw Stability An aliquot of AB0089 (20 mg/ml in CST pH 7.0) was placed in a styrofoam container (to slow down the freezing rate) and the container placed at -80°C. Freeze and thaw six times. AB0089 showed no loss of monomer by SEC after 6 cycles of freeze/thaw. Protein concentrations as measured by A280 remained the same (Figure 183 and Table 177).

No loss of purity was detected by reducing or non-reducing CE-SDS after 6 freeze/thaw cycles (Figure 184 and Table 178).

Agitation AB0089 (10 mg/ml in 20 mM sodium citrate, 250 mM sucrose, pH 7.0, with or without 0.01% Tween®-80) was shaken at 300 rpm in deep well plates at room temperature for 3 days. . There was no loss of monomer detected by SEC (Figure 185 and Table 179), no loss of protein concentration (A280), and no increase in turbidity (A340) after agitation stress.

High Concentrations AB0089 was concentrated to approximately 10, 15, 25, 50, 100, 150 and 175 mg/mL (pH 7.4) in PBS and recovery was monitored by visual inspection, concentration (A280), and SEC. For SEC analysis, samples were injected undiluted (injection volumes were normalized to concentration). AB0089 could be concentrated to greater than 175 mg/ml with minimal percent monomer loss (Figure 186, Figure 187, Table 180) and no visible precipitation.

Low pH Hold Two different methods were used to assess the behavior of AB0089 during low pH hold (simulated virus inactivation). The first method mimicked the manufacturing process in which the eluate from the first chromatography step (Protein A) was held at low pH prior to further purification. In the second method, fully purified AB0089 was incubated under low pH conditions. This method was used to assess potential low pH-induced chemical modifications of CDRs.

Low pH Hold Exposure of Protein A Eluate To determine if AB0089 tolerated low pH hold, AB0089 Protein A eluate was adjusted to pH 3.5 and held at room temperature for 2 hours. . After the holding period, the protein A eluate was neutralized with 1.0 M Tris, pH 8.3 to achieve neutral pH. Analytical SEC was performed to see if there was a change in profile or aggregate content before and after low pH exposure (Figure 188). No change was observed in the SEC profile of AB0089 after the low pH hold compared to the "no hold" control sample, thereby demonstrating that AB0089 is suitable for the low pH hold/virus inactivation conditions used in the manufacture of biologics. suggests that it is very stable during

The protein was further processed in the second step of the DF platform purification method and analyzed using an additional panel of assays compared to purified protein that was not subjected to low pH holding. Chemical modifications of amino acid side chains can usually be observed globally using cIEF. The cIEF profiles of the AB0089 control and the low pH hold lot are visually very similar, with relative quantification of acidic, major, and basic species all within 5% of each other. This indicates that low pH retention had no measurable effect on the charge profile of AB0089 (Figure 189 and Table 181).

The affinity of AB0089 for hCLEC12a, hNKG2D, and CD16aV was not affected by low pH holding (Figure 190 and Table 182). No difference in active protein content was observed. Differences in binding affinities and maximal binding responses between control and low pH holding samples were within the experimental variation of the assay. This indicates that AB0089 retains affinity for all three targets after low pH holding.

The efficacy of AB0089 and control samples subjected to low pH holding was tested against CLEC12A+HL-60 cancer cells in the KHYG1-CD16aV bioassay. The potency of AB0089 did not change after the low pH holding step (Figure 191 and Table 183).

のＤＴモチーフがウイルス不活化ステップの間に異性化するか否かを調査するために、追加の低ｐＨ保持研究を実施した。次の手順を使用して、完全に精製されたＡＢ００８９材料から低ｐＨ保持材料を調製した。
１．ＡＢ００８９は、ＰｒｏＡ溶出を模倣するために０．１ｍグリシン、ｐＨ ３．０を使用して１：１に希釈した
２．２ｍ酢酸でｐＨを３．５±０．１に調整した（低ｐＨ保持条件を模倣するため）
３．低ｐＨ保持は室温で６０分間行った。
４．１ｍのＴｒｉｓ、ｐＨ８．０を用いてｐＨを約７．５に中和した
５．ＡＢ００８９をＰＢＳ、ｐＨ７．４に緩衝液交換した Low pH retention exposure of fully purified protein CDRH3 of CLEC12A-targeted scFv

An additional low pH holding study was performed to investigate whether the DT motif of .RTM. A low pH retention material was prepared from fully purified AB0089 material using the following procedure.
1. AB0089 was diluted 1:1 using 0.1 m glycine, pH 3.0 to mimic ProA elution pH was adjusted to 3.5 ± 0.1 with 2.2 m acetic acid (low pH retention to mimic conditions)
3. A low pH hold was performed at room temperature for 60 minutes.
4. Neutralized pH to about 7.5 using 1 m Tris, pH 8.0. AB0089 was buffer exchanged into PBS, pH 7.4

The final low pH retention material (AB0089 lot AB0089-004) was analyzed using a panel of biological assays: SEC, cIEF, SPR binding to CLEC12A, peptide map, and potency to control (AB0089 lot AB0089-002). ). Analytical SEC was performed to determine if there was a change in profile or aggregate content before and after low pH exposure (Figure 192 and Table 184). No change in the % monomer profile was observed after the low pH hold.

As seen in the first low pH hold study, the cIEF profiles of the AB0089 control and the low pH hold lot were visually very similar, and the relative quantification of acidic, major, and basic species were all consistent with each other. is within 6% of Therefore, low pH retention did not significantly affect the charge profile of AB0089 (Figure 193 and Table 185).

The affinity of AB0089 for hCLEC12a was unchanged compared to controls (Figure 281 and Table 186). Differences in binding affinities and maximal binding responses between control and low pH holding samples are within the experimental variation of the assay. This indicates that the low pH incubation had no effect on the binding of the CLEC12A binding arm to the target antigen.

The efficacy of AB0089 after low pH hold against CLEC12A+HL-60 cancer cells was evaluated with the KHYG1-CD16V bioassay. No difference was observed between low pH retention and efficacy of control samples (Figure 282 and Table 187).

To determine whether low pH retention induced changes in CDR modifications (alterations), control and low pH retention samples were analyzed by LC-MS/MS peptide mapping. Based on the peptide map data, all CDRs of AB0089 are resistant to modification (alteration) during low pH holding (Table 188). No evidence of aspartic isomerization was observed based on manual inspection of peak shape.

Taken together, these results indicate that AB0089 is an improved version of AB0192 with an optimized CMC profile. The molecular format and structure of AB0089 fulfills conceptual requirements for new multispecific therapeutics that simultaneously engage CLEC12A, NKG2D, and CD16a to treat AML. AB0089 is potent and has a favorable developability profile with no observed sequence liability or significant post-translational modifications under any stress. AB0089 binds to an epitope on CLEC12a similar to tepositamab (Merus).

Example 18. AB0089 Cytotoxicity and Mechanism of Action Objective To characterize the mechanism of action of AB0089, measure the activity of AB0089 in an in vitro cell-based assay using physiologically relevant conditions.

Study Design The primary mechanism of action of AB0089 was tested using an in vitro assay system with human NK cells and CLEC12A+ cancer cells. Other effector functions besides NK cell-mediated cytolysis were also combined with CLEC12A+ cancer cells to test additional mechanisms of action of AB0089. Macrophages are the effector cells in the ADCP assay and human serum was used as the source of complement to test CDC activity.

Materials and Methods Primary Cells and Cell Lines Human cancer cell lines were obtained from research cell banks. The following cell lines (item numbers; suppliers) were used: PL21 (ACC-536; DSMZ) and HL60 (CCL-240; ATCC).

Human blood was obtained from Biological Specialty Corporation (225-11-04). PBMCs were isolated by density gradient centrifugation and NK cells were purified using negative depletion with magnetic beads (StemCell Catalog #17955). The manufacturer's instructions were followed for the purification of NK cells from PBMC. Frozen NK cells were purchased from BioIVT HUMAN-HL65-U-200429, CD14+ monocytes were obtained from BioIVT (HMN370652, HMN370653, HMN370654) and frozen PBMC were purified in-house (see above) for later use. frozen to

Cell culture materials The following items (suppliers; item numbers) were used: RPMI-1640 (Corning; 10040CV), DMEM (Corning; 10031CV), heat-inactivated FBS (ThermoFisher; 16140-071), GlutaMax I (ThermoFisher 35050-061), penn/strep (penicillin/streptomycin) (ThermoFisher; 151450-122), 2-mercaptoethanol (MP Biomedicals; 02194705), IL-2 (PeproTech; 200-02), CFSE (ThermoFisher; C34554) , Cell Trace Violet (ThermoFisher; C34557), rhM-CSF (R & D Systems; 216-MC/CF), TrypLE (ThermoFisher; 12604-013), BFA (Biolegend; 420601), Monensin (BioLegend; 42) Human serum (Sigma Aldrich; S1764-5X1ML).

Assay Materials The following items (supplier; item number) were used: Hepes buffered saline (Alfa Aesar; J67502), BATDA reagent (Perkin Elmer; C136-100), TC round bottom 96-well plates (Corning; 3799). , Europium solution (Perkin Elmer; C135-100), DELFIA yellow microplate 96-well (Perkin Elmer; AAAAND-0001), fixation buffer (BioLegend; 420801), Zombie NIR (BioLegend; 77184), and anti-MHC class I clone. W6/32 (Invitrogen; HLA-C1).

The following test substances (description) were used: AB0237 (CLEC12A targeting TriNKET (A49) with Fab NKG2D binding and scFv CLEC12A binding domain (1602)), AB0300 (AB0237 with LALAPG mutation in CH2 domain), AB0037 mAb ( humanized 1602 anti-CLEC12A mAb), AB0089 (ClEC12A-targeted TriNKET with Fab NKG2D binding (A49) and scFv CLEC12A binding domain (1739)), AB0305 mAb (humanized 1739 anti-CLEC12A mAb), AB0192 (Fab CLEC12A-targeted TriNKET (A49) with Fab NKG2D binding, and scFv CLEC12A binding domain (1797)), AB0306 mAb (humanized 1797 anti-CLEC12A mAb), and F3'-palivizumab (non-targeted TriNKET with Fab NKG2D binding (A49), and palivizumab scFv binding domain from ).

The following instruments (serial numbers) were used: Attune NxT (2AFC228271119), BD Celesta (H66034400085), BD Celesta (H66034400160), Spectramax i3x (363704226), and Spectramax i3x (363704226).

Preparation of Reagents RPMI primary cell culture medium was prepared starting with RPMI-1640. 10% HI-FBS, 1x GlutaMax, 1x penn/strep (penicillin/streptomycin), and 50 μM 2-mercaptoethanol were added for complete medium. RPMI cell culture medium was prepared starting with RPMI-1640. 10% HI-FBS, 1x GlutaMax, and 1x penn/strep (penicillin/streptomycin) were added for complete medium. 1xHBS was prepared by diluting 1 part 5xHBS into 4 parts DI water. The solution was sterile filtered before use.

DELFIA Cytotoxicity Assay Target Cell Labeling:
Briefly, CLEC12A+ target cells were pelleted and washed with 1×HBS. Target cells were resuspended at 10 ⁶ cells/mL in prewarmed RPMI primary cell culture medium. BATDA reagent (bis(acetoxymethyl)2,2':6',2''-terpyridine-6,6''-dicarboxylate) was diluted 1:400 into the cell suspension. Cells were mixed and incubated for 15 minutes at 37°C with 5% _CO2 . Labeled target cells were washed 3 times with 1×HBS and resuspended at 5×10 ⁴ cells/mL in RPMI primary cell culture medium.

Assay plate preparation:
Resting human NK cells were removed from culture, pelleted, and cells were resuspended at 0.5×10 ⁶ cells/mL in RPMI primary cell culture medium. Quadruple test substances were prepared in RPMI primary cell culture medium. To a round-bottom TC96-well plate was added 100 μl of labeled target cells, 50 μl of 4xTriNKET/mAb, and 50 μl of effector cells. Control wells for background were prepared by pelleting labeled target cells and 100 μl of supernatant containing 100 μl of RPMI primary cell culture medium was added to background wells. Spontaneous release wells were prepared by adding 100 μl of labeled target cells to wells containing 100 μl of RPMI primary cell culture medium. Maximum release wells were prepared by adding 100 μl of labeled target cells to wells containing 80 μl of RPMI primary cell culture medium and 20 μl of 10% Triton® X-100 solution. Assay plates were incubated at 37° C., 5% CO ₂ for 2-3 hours.

Assay plate reading:
The assay plate was removed from the incubator and the plate was centrifuged to pellet the cells. 20 μl of supernatant was removed from each well and transferred to a clean 96-well yellow DELFIA assay plate. 200 μl of room temperature europium solution was added to each well and the plate was placed on a plate shaker at 250 RPM for 15 minutes. The plate was read using a SpectraMax i3x HTRF cartridge with the following PMT and optical settings.
● Integration time 0.4ms
●Excitation time 0.05ms
●Number of pulses 100
●Measurement delay 0.25ms
●Reading height 2.36mm

Data analysis:
An average of background samples was calculated and subtracted from all samples. Specific lysis was calculated as follows:
% specific lysis = (sample-spontaneous)/(maximum-spontaneous)*100%

Data were fitted to a four-parameter nonlinear regression model using Prism GraphPad 7.0 or 8.0.

IFNγ and CD107a Activation Assay Plate Setup:
A human cancer cell line expressing CLEC12A was harvested from culture and cells were adjusted to 2×10 ⁶ cells/mL. Test substances were diluted in culture medium. Resting NK cells or PBMC were harvested from culture, cells were washed and resuspended in culture medium at 2-4×10 ⁶ cells/mL. IL-2 and fluorophore-conjugated anti-CD107a were added to NK cells or PBMCs for activation culture. Brefeldin-A (BFA) and monensin were diluted in culture medium to block protein transport from cells for intracellular cytokine staining. In a 96-well plate, 50 μl of tumor targets, mAbs/TriNKETs, BFA/monensin, and NK cells were added for a total culture volume of 200 μl. After the plates were incubated for 4 hours, samples were prepared for FACS analysis.

Sample preparation for FACS:
After 4 hours of activation culture, cells were prepared for analysis by flow cytometry using the fluorophore-conjugated antibodies listed in Table 189. CD107a and IFNγ staining were analyzed in the CD3-CD56+ population to assess NK cell activation.

Cells of interest were identified using an FSC vs. SSC plot and an appropriately shaped gate was drawn around the cells. Doublet cells were eliminated by displaying FSC-H versus FSC-A plots within the gating cells. Live cells were gated within the single cell population. Within viable cells, NK cells were identified as CD56+CD3-. CD107a degranulation and intracellular (IC) IFNγ were analyzed within the NK cell population.

ADCP Assay Primary human CD14+ monocytes are obtained from BioIVT. Monocytes were isolated from peripheral blood by negative selection with yields >93% pure. Monocytes are cultured with 50 ng/ml rhM-CSF (R&D Systems) for 6 days and replenished with rhM-CSF every 2 days. After 6 days, macrophages were detached from the culture flask(s) with TrypLE Express (Gibco) and labeled with Celltrace Violet (2 μM, ThermoFisher). Target cells were labeled with CFSE (2 μM, ThermoFisher). Target cells (1×10 ⁴ cells/well) are first incubated with the indicated concentrations of each test substance for 30 minutes in round-bottomed 96-well plates. The indicated macrophages (8×10 ⁴ cells/well) were then added to the appropriate wells and the plates were incubated for an additional 2 hours. Cells were stained with Zombie NIR dye (Biolegend) for live/dead discrimination and fixed. Phagocytosis is quantified by Celesta HTS (BD Biosciences) using FlowJo 10.5.3 data analysis. Data were gated on single viable cells and % phagocytosis was calculated as follows: % phagocytosis = 100 x (CFSE+, Celltrace Violet+ cell counts)/(CFSE+ cell counts). Data were graphed and analyzed using GraphPad Prism.

CDC Assay CDC was measured with the DELFIA cytotoxicity assay. PL21 cancer cell targets were labeled with a BATDA labeling reagent. Labeled target cells were then incubated with the indicated concentrations of TriNKET or mAbs for 30 minutes to allow opsonization. Human serum was added to a final concentration of 5%. The plate was then incubated for 60 minutes before the assay was terminated. 20 μl of culture supernatant was transferred to DELFIA yellow plates. 200 μl of europium solution was added per well and incubated for 15 minutes at room temperature with shaking in the dark. Fluorescence was then measured on a Spectramax plate reader.

Results and Discussion Resting Human NK Cell Lysis of CLEC12A+ Target Cells The activity of AB0089 and AB0192 was characterized using resting primary human NK cells from healthy donors in co-culture with either human CLEC12A+ AML lines PL21 or HL60. was taken. AB0089 and AB0192 were compared to parental hIgG1 mAbs AB0305 and AB0306, respectively. Figure 194 shows representative plots of PL21 leukemia target cell lysis by resting human NK cells in the presence of AB0089. AB0089 exhibited a dose-dependent cytolytic enhancing activity that was more potent and reached a higher maximal lysis than the parent mAb AB0305. Results of a similar cytotoxicity assay using NK cells from a total of 6 donors are summarized in Table 190. Across the assay, the potency of AB0089 to enhance NK cell lysis of PL21 target cells had a mean EC50 value of 0.25±0.24 nM.

Figure 195 shows representative plots of HL60 leukemia target cell lysis by resting primary human NK cells in the presence of AB0089. Again, AB0089 showed overall stronger dose-dependent lysis and higher specific lysis than its parent mAb AB0305. Assay results using NK cells from four healthy donors yielded a mean EC50 of 0.09±0.05 nM, as summarized in Table 191.

Figure 196 shows representative plots of PL21 leukemia target cell lysis by resting human NK cells in the presence of AB0192. AB0192 exhibited a dose-dependent cytolytic enhancing activity that was more potent and reached a higher maximal lysis than the parent mAb AB0306. Results of a similar cytotoxicity assay using NK cells from a total of 6 healthy donors are summarized in Table 192. Across the assay, the potency of AB0192 to enhance NK cell lysis of PL21 target cells had a mean EC50 value of 0.61±0.60 nM.

Figure 197 shows representative plots of HL60 leukemia target cell lysis by resting human NK cells in the presence of AB0192. Again, AB0192 showed overall stronger dose-dependent lysis and higher specific lysis than its parent mAb. Assays using NK cells from four healthy donors resulted in a mean EC50 of 0.30±0.27 nM, as summarized in Table 193.

AB0089 Activity in the Presence of Soluble MIC-A We also tested the activity of AB0089 in the presence of soluble NKG2D ligands. We chose to use a recombinant version of the NKG2D ligand MIC-A in these assays. MIC-A exhibits broad expression across cancer indications and is known to shed from the cell surface and accumulate in patient sera. Soluble MIC-A-Fc was added to the NK cell cytolytic assay system at 20 ng/mL, a physiologically relevant serum concentration found in cancer patients. Figure 198 shows dose response curves of AB0089 and its parent mAb AB0305 in primary NK cell cytolytic assay against PL21 target cells in the absence and presence of soluble MIC-A. Addition of MIC-A did not affect potency or maximal dissolution achieved by AB0089 (see Table 194 for EC50 and maximal dissolution values).

AB0089 Stimulation of NK Cell IFNγ Production and Degranulation In addition to direct lysis of target cells, NK cells also produce cytokines upon activation. Therefore, we wanted to investigate alternative readouts of NK activation in addition to target cell lysis. We chose to analyze IFNγ production and CD107a degranulation from NK cells co-cultured with CLEC12A+ AML cells in the presence of AB0089. Resting NK cells within PBMCs in co-culture with PL21 and HL60 target cells showed little basal induction of CD107a degranulation or intracellular IFNγ accumulation after 4 hours (Figures 199 and 200, respectively). Addition of AB0089 to co-cultures resulted in strong induction of degranulation and IFNγ production in a dose-response manner. In contrast, neither the parental mAb AB0305 nor TriNKETF3′-palivizumab, which does not target CLEC12A, showed a robust expansion of CD107a+IFNγ+NK cells. Assays were performed using three independent PBMC donors in co-culture with PL21 or HL60 target cells; the results are summarized in Tables 195 and 196, respectively.

AB0089 Induces ADCP Activity The Fc domain of human IgG1 antibodies can mediate three different types of effector functions.

One type of Fc-mediated effector function is ADCC, which is carried out by engagement of CD16 on NK cells; NK cell stimulation has been extensively characterized for AB0089. A second Fc-mediated effector function is ADCP, in which macrophages attack and engulf antibody-coated cells. For AB0089 and the alternative CLEC12A TriNKET, we wanted to assess its ability to induce ADCP of opsonized target cells. As effector cells, an in vitro assay system was utilized using M0 macrophages obtained from culturing purified CD14+ monocytes with M-CSF. CLEC12A+ target cells were labeled with Cell Trace CFSE dye, opsonized with test substances, and co-cultured with Cell Trace Violet-labeled MO macrophages. Phagocytosis was analyzed by flow cytometry as Cell Trace Violet + Cell Trace CFSE + (double positive) events.

AB0089 and the other CLEC12A TriNKETs AB0237 and AB0192 all enhanced phagocytosis of PL21 AML cells by M0 macrophages. All of the parental mAbs of each TriNKET also showed the ability to induce M0 macrophage phagocytosis of opsonized target cells, although at lower maximal levels compared to their respective TriNKET counterparts (Figure 201). AB0237si carrying a mutation in the CH2 domain to silence Fcγ receptor binding served as a negative control As expected, AB0237si was unable to mediate ADCP of opsonized target cells. Results using M0 macrophages from 3 different donors are summarized in Table 197.

AB0089 lacks CDC activity A third effector function of human IgG1 isotype antibodies is the initiation of the complement cascade, causing complement dependent cytotoxicity (CDC). AB0089 is constructed using a human IgG1 Fc domain; therefore, we wanted to understand AB0089's ability to stimulate CDC activity. PL21 cells were used as targets and 5% human serum as the source of complement factors. Neither AB0089 nor its parent mAb AB0305 stimulated complement-mediated killing of PL21 AML cells (FIG. 202). In contrast, a positive control antibody against MHC class I (clone W6/32)) showed dose-dependent lysis of PL21 target cells in the presence of human serum, confirming that serum has active complement factors. ing. Cell characterization analysis showed similar expression levels of CLEC12A and MHC class I on PL21 target cells.

AB0089 showed potent activity in a variety of physiologically relevant contexts. AB0089 was first tested in a CLEC12A+ target cell killing assay using resting primary human NK cells. The inventors chose to test killing of CLEC12A+ target cells from AML, which have been shown to overexpress CLEC12A in the clinic. In these experiments, HL60 and PL21 were chosen as representative AML cell lines.

AB0089 enhanced primary NK cell cytolysis of both target cell lines and outperformed its parent hIgG1 antibody in enhancing cytolysis. We also examined the effect of soluble NKG2D ligands on the activity of AB0089 and found that physiological levels of soluble MIC-A-Fc did not alter the activity of AB0089 in cell lysis.

In addition to cytolysis, AB0089 also stimulated cytokine production from NK cells co-cultured with CLEC12A+ target cells. AB0089 enhanced intracellular accumulation of IFNγ and degranulation of NK cells in co-cultures of human PBMCs with PL21 and HL60 AML cells. EC50 values for IFNγ and CD107a induction by AB0089 were consistent with EC50 values generated in cytolytic assays.

Finally, additional mechanisms of action of AB0089 were investigated. AB0089 was demonstrated to engage macrophages to mediate ADCP of AB0089-opsonized target cells. In contrast, AB0089 does not mediate CDC of AB0089-opsonized target cells.

AB0089 was demonstrated to have potent activity against CLEC12A+ cancer cells through various effectors such as NK cells and macrophages. Furthermore, the activity of AB0089 was maintained in the presence of physiologically relevant concentrations of the soluble NKG2D ligand MIC-A.

Example 19. AB0089 Binding to CLEC12A+ Cell Lines and Primary AML Purpose To determine the binding of AB0089 to human CLEC12A-expressing cells. Quantify CLEC12A surface retention after incubation with AB0089. Binding of AB0089 to primary AML patient samples is shown and compared to CLEC12A expression patterns.

Study Design A representative cell line positive for CLEC12A surface expression was used to characterize AB0089 cell binding. Primary AML patient samples were obtained from Creative Bioarray and BioIVT. Binding of AB0089 and the parental mAb AB0305 was compared to the commercially available CLEC12A antibody.

Materials and Methods Primary Cells and Cell Lines Human cancer cell lines were obtained from research cell banks. The following cell lines (item numbers; suppliers) were used: PL21 (ACC-536; DSMZ), HL60 (CCL-240; ATCC), Ba/F3-hCLEC12A (manufactured in-house) and Ba/F3 (DSMZ ACC- 300).

Human CLEC12A was introduced into Ba/F3 parental cells by retroviral transduction.

Cell culture materials The following items (suppliers; item numbers) were used: RPMI-1640 (Corning; 10040CV), DMEM (Corning; 10031CV), heat-inactivated FBS (ThermoFisher; 16140-071), GlutaMax I (ThermoFisher 35050-061), penn/strep (penicillin/streptomycin) (ThermoFisher; 151450-122), and 2-mercaptoethanol (MP Biomedicals; 02194705).

Assay Materials The following items (supplier; item number) were used: Hepes buffered saline (Alfa Aesar; J67502), BATDA reagent (Perkin Elmer; C136-100), TC round bottom 96-well plates (Corning; 3799). , Europium solution (Perkin Elmer; C135-100), and DELFIA yellow microplate 96-well (Perkin Elmer; AAAAND-0001).

The following test substances (description) were used: AB0237 (ClEC12A-targeted TriNKET (A49) with Fab NKG2D binding and scFv CLEC12A binding domain (1602)), AB0300 (AB0237 with LALAPG mutation in CH2 domain), AB0237 -Dead-2D (AB0237, NKG2D binding domain mutated to eliminate binding), AB0037 mAb (humanized 1602 anti-CLEC12A mAb), AB0089 (CLEC12A targeting TriNKET with Fab NKG2D binding (A49) and scFv CLEC12A binding domain (1739)), AB0305 mAb (humanized 1739 anti-CLEC12A mAb), AB0192 (ClEC12A-targeted TriNKET with Fab NKG2D binding (A49), and CLEC12A binding domain of scFv (1797)), AB0306 mAb (humanized 1797 anti-CLEC12A mAb), hcFAE-A49. h6E7 (CLEC12A-targeted TriNKET (A49) with Fab NKG2D binding, and Fab CLEC12A binding domain (Genentech 6E7)) and hcFAE-A49. CLL1-Merus (ClEC12A-targeted TriNKET (A49) with Fab NKG2D binding, and Fab CLEC12A binding domain (Merus CLL-1 binder)).

The following instruments (serial numbers) were used: Attune NxT (2AFC228271119), BD Celesta (H66034400085), and BD Celesta (H66034400160).

Preparation of Reagents RPMI primary cell culture medium was prepared starting with RPMI-1640. 10% HI-FBS, 1x GlutaMax, 1x penn/strep (penicillin/streptomycin), and 50 μM 2-mercaptoethanol were added for complete medium. RPMI cell culture medium was prepared starting with RPMI-1640. 10% HI-FBS, 1x GlutaMax, and 1x penn/strep (penicillin/streptomycin) were added for complete medium. 1xHBS was prepared by diluting 1 part 5xHBS into 4 parts DI water. The solution was sterile filtered before use.

Detection of binding and surface binding mAb/TriNKET
For FACS staining, 100,000 cells were seeded per well in 96-well plates. Cells were washed with PBS. Cells were incubated in a 1:2000 dilution of live/dead dye in PBS for 15 minutes and then washed with FACS buffer. TriNKET or mAbs were diluted in FACS buffer and 50 μl containing diluted TriNKET or mAb was added to the cells. Cells were incubated on ice for 20 minutes. After incubation, cells were washed with FACS buffer. Anti-human IgG-Fc secondary antibody was diluted in FACS buffer and 50 μl added per well to detect bound TriNKET or mAb. Cells were incubated on ice for 20 minutes and then washed with FACS buffer. 50 μl of Fixation Buffer was added to each well and the cells were incubated for 10 minutes at room temperature. Cells were washed with FACS buffer and resuspended in FACS buffer for analysis on BD FACS Celesta SN#H66034400085 or BD FACS Celesta SN#H66034400160. Cells of interest were identified using an FSC vs. SSC plot and an appropriately shaped gate was drawn around the cells. Doublet cells were eliminated by displaying FSC-H versus FSC-A plots within the gated cells. Live cells were gated within the single cell population. Within the live gate, the mean fluorescence intensity (MFI) was calculated for each sample and the second order only control. Background fold (FOB) was calculated as the ratio of test article MFI to second-order only background MFI. Data were fitted to a four-parameter nonlinear regression model using GraphPad Prism 7.0.

Surface Retention Assay For FACS staining, 100,000 cells per well were plated in duplicate in 96-well plates. Cells were washed with FACS buffer. Test substances were diluted to 40 nM and added to each of the duplicate plates. The first plate was incubated on ice for 2 hours and the second plate was moved to 37°C for 2 hours. After incubation, cells were washed with PBS and cells were incubated in a 1:2000 dilution of live/dead dye in PBS for 15 minutes before washing with FACS buffer. Anti-human IgG-Fc secondary antibody was diluted in FACS buffer and 50 μl added per well to detect bound TriNKET or mAb. Cells were incubated on ice for 20 minutes and then washed with FACS buffer. 50 μl of Fixation Buffer was added to each well and the cells were incubated for 10 minutes at room temperature. Cells were washed and resuspended in FACS buffer for analysis on BD FACS Celesta SN#H66034400085 or BD FACS Celesta SN#H66034400160. Cells of interest were identified using an FSC vs. SSC plot and an appropriately shaped gate was drawn around the cells. Doublet cells were eliminated by displaying FSC-H vs. FSC-A plots within the gated cells. Live cells were gated within the single cell population. Within the live gate, the MFI for each sample was calculated. The hIgG1 isotype control MFI was subtracted from all test article MFIs. Surface retention was then calculated as follows:
% surface retention = (2 hours 37°C MFI/2 hours ice MFI) * 100

Analysis of AB0089 Binding in Primary AML Samples Frozen AML patient samples were thawed at 37° C. and used immediately in binding assays. Test article, hIgG1 isotype control and commercial reagent anti-CLEC12A antibody clone 50C1 were labeled using the Biotium Mix-n-Stain CF568 labeling kit. Manufacturer's instructions were followed. AML samples were stained with a cocktail of antibodies as described in Tables 198 and 199. Samples were analyzed using an Attune NxT cytometer equipped with a CytKick Max autosampler. Raw data were analyzed using FlowJo X software.

Results and Discussion CLEC12A TriNKET binds to cell lines expressing human CLEC12A The binding of CLEC12A TriNKET and its parent mAb was tested using three cell lines expressing human CLEC12A (Figure 203). PL21 and HL60 are human AML cell lines with endogenous CLEC12A expression, whereas mouse Ba/F3 cells were engineered to express human CLEC12A.

As comparators, two tools TriNKET were generated using the CLEC12A binding domains of Genentech (h6E7) and Merus (CLL1-Merus) known in the public domain. Duobody TriNKET with Fab CLEC12A binding domain was paired with FabNKG2D binder A49. Duobody TriNKET hcFAE-A49. h6E7 is derived from a Genentech binder and hcFAE-A49. CLL1-Merus is derived from the Merus binder.

The lead TriNKETAB0089 and the alternate CLEC12A TriNKETs AB0237 and AB0192 all bound to CLEC12A-expressing cells with nanomolar potency (Table 4). AB0089 had reduced potency but reached maximum FOB compared to the parental mAb AB0305 in the three cell lines tested. AB0237 exhibited comparable binding to its parental mAb AB0037, with potency values within 2-fold of each other on all three cell lines. AB0237 variants AB0237-Dead 2D and AB0300, which has mutations in the NKG2D and CD16 binding domains, respectively, showed similar cell binding compared to AB0237. In contrast, AB0192 showed reduced potency and maximum FOB compared to its parent mAb AB0306.

Compared to the tool TriNKET, AB0089 is more efficient than hcFAE-A49. Comparable cell loading to CLL1-Merus, and hcFAE-A40. It showed a higher cell load than h6E7. Furthermore, AB0237 binding to the CLEC12A cell line was associated with hcFAE-A49. It was more potent than CLL1-Merus and comparable in potency to hcFAE-h6E7. All EC50 binding potencies and maximum FOB binding are summarized in Table 201.

CLEC12A-TriNKET Surface Retention Internalization of CLEC12A after antibody ligation has been described in the literature. This phenomenon has been exploited to develop CLEC12A-targeted antibody-drug conjugates, which rely on internalization of the conjugated drug to kill the target cell. However, TriNKET activity requires cell surface retention of CLEC12A in complex with TriNKET to allow NK cell engagement and effector activity on CLEC12A+ cancer cells. To better understand the retention of CLEC12A on the cell surface in the presence of CLEC12A TriNKETs, we investigated the effect of the retention of CLEC12A on the cell surface on the CLEC12A+ cell line after 2 hours of incubation at 37°C compared to incubation on ice. binding of saturating concentrations of each TriNKET and parental mAb.

More than 90% of cell surface CLEC12A was retained by each TriNKET when bound to cells for 2 hours at 37°C. Figure 204 shows examples of surface retention of AB0089, AB0192, AB0237 and their parental antibodies in HL60 and PL21 AML strains. Data from three independent experiments are summarized in Table 202. AB0089 appeared to exhibit superior surface retention compared to other CLEC12A TriNKETs and their parent mAbs.

However, AB0089 showed a lower binding signal after incubation on ice compared to AB0237, but a similar signal after 2 h incubation at 37 °C, possibly inflating the surface retention value of AB0089 ( Figure 205). Nevertheless, these data suggest a temperature-dependent binding of AB0089 to reach equilibrium, which could be attributed to a two-step binding mechanism suggested by the kinetic binding data. There is (Example 17).

In contrast, AB0192 and its parental mAb AB0306, derived from different parental murine mAbs, showed similar surface retention on HL60 and PL21 cells. These data indicate that CLEC12A surface retention depends on whether it binds monovalently (as in the case of F3'TriNKET) or bivalently (as in mAb) and the epitope of the particular binder. is shown to obtain

AB0089 Binds to CLEC12A in Human AML Patient Samples Primary bone marrow and PBMC samples from a total of 10 AML patients were obtained (see Table 200 for sample information) and the binding of AB0089 and its parent mAb AB0305 to primary AML cells. investigated the bond. The binding patterns of fluorophore-labeled test substances AB0089 and AB0305 were compared with the commercially available anti-CLEC12A antibody clone 50C1 (Lahoud et al, The Journal of Immunology. 2009; 182:7587-7594). Among the 10 donors, the percentage of CLEC12A+ cells within the AML blast gate varied widely from approximately 40-95%. AML patient samples from different FAB subtypes were analyzed, which may contribute to the variation in CLEC12A expression and observed proportions.

AB0089 and AB0305 show binding patterns comparable to anti-CLEC12A clone 50C1 across the 10 AML patient samples tested (Figure 206). Blasts were also gated to examine the leukemic stem cell-enriched CD34+CD38− subpopulation from each sample. Repeatedly, AB0089 and AB0305 stained similarly with CD34+CD38- blasts as clone 50C1 (Fig. 207). Altogether, these data confirm that AB0089 can bind CLEC12A on CD34+CD38- subsets enriched for primary AML blasts and LSCs.

AB0089 and other CLEC12A TriNKETs showed similar nanomolar potency across lines against Ba/F3 cells ectopically expressing human CLEC12A and PL21 and HL60 AML cells expressing endogenous CLEC12A in a dose-responsive manner. coupled to Despite literature reports of CLEC12A internalization after antibody ligation, both AB0089 and the alternative CLEC12A TriNKETs AB0237 and AB0192 showed high cell surface retention of CLEC12A after 2 hours of incubation with HL60 and PL21 CLEC12A+ cell lines. . Finally, AB0089 and its parent mAb AB0305 bound similarly to the commercial reagent anti-CLEC12A antibody clone 50C1 in bulk and to CD34+CD38- blasts across primary AML patient samples from 10 donors. Taken together, these data support the strong binding of AB0089 to cells expressing CLEC12A and indicate that AB0089 binds to primary AML blasts.

Example 20. AB0089 CLEC12A TriNKET
Purpose The purpose of this study is to describe the molecular format, design, structure and characterization of CLEC12A TriNKET AB0192. Specifically, in this report, we a) describe the molecular format and design of AB0192, b) provide information on the expression and purification of AB0192, and c) provide basic biochemical and biophysical understanding of the molecule. d) determine the affinity of AB0192 for recombinant human targets (CLEC12A, NKG2D and CD16a); e) bind AB0192 to isogenic and AML cancer cell lines expressing human CLEC12A. f) demonstrate the selectivity of AB0192; g) determine the potency of AB0192 in killing AML cancer cells; h) assess binding epitopes; i) assess binding to FcγR and FcRn; Describe the behavior of AB0192 in feasibility studies, including j) accelerated stability, forced degradation, and manufacturability, compared to controls.

Study Design A mouse anti-human CLEC12A mAb (9F11.B7) was discovered by immunizing mice. 9F11. B7 was humanized, converted to scFv and combined with A49M-I NKG2D Fab binding arms to generate F3'-TriNKET (AB0192). AB0192 was evaluated by binding to recombinant human and cyno hCLEC12A, binding to isogenic and cancer cells expressing CLEC12A, and potency of the molecule against CLEC12A+ AML cancer cells. Biophysical and biochemical properties of AB0192 were determined, including thermal stability (DSC), hydrophobicity (HIC), and pI (cIEF). Specificity of binding was assessed by PSR, binding to cells not expressing the target of interest, and HuProt™ arrays. AB0192 was expressed in two different mammalian cell lines (Expi293 and ExpiCHO) and purified using the Dragonfly Tx2 step platform purification method. AB0192, including CLEC12A, NKG2D, CD16a (V158 and F158), an expanded panel of Fcγ receptors (CD32a (H131 and R131), CD16b, CD32b, cynomolgus CD16, human and cynomolgus CD64), and FcRn (human and cynomolgus) ) has been fully characterized. Binding epitopes were characterized in binning experiments with CLEC12A binding molecules from Merus and Genentech. Finally, AB0192 was tested in the Dragonfly Tx platform formulation (20 mM histidine, 250 mM sucrose, 0.01% Tween®-80, pH 6.0) and 20 mM citrate, 250 mM sucrose, 0.01% Tween® )-80, accelerated (40° C.) stability in pH 7.0 buffer, forced degradation (long-term pH 5, pH 8 stress, forced oxidation), and manufacturability (freeze/thaw, agitation, low pH holding). Evaluated with a full feasibility panel, including:

Materials and Methods The methods used to prepare and characterize AB0192 are similar to those used in Example 17.

Test Article AB0192-002, (benchmarking record name AB0192), humanized TriNKET expressed in Expi293 (lot AB0192-002).

AB0192-005, (benchling record name AB0192), humanized TriNKET expressed in ExpiCHO (lot AB0192-005).
Protein reagent

Results Discovery of AB0192TriNKET Hybridoma subclone 9F11. B7 was discovered through immunization of the BALB/c mouse strain with recombinant human CLEC12A-his performed at Green Mountain Antibodies (Burlington, Vt.).

Mouse 9F11. B7 was humanized by grafting mouse CDRs into appropriate human frameworks (VH3-30*02 and VK1-33) without loss of affinity for recombinant CLEC12A or cell surface expressed targets (Fig. 208). ). Seven mouse backmutations were introduced to maintain the CDR architecture. Humanized 9F11. B7 mAb was converted to scFv and paired with NKG2D-targeting A49 MI Fab to generate F3'TriNKET (AB00192). This TriNKET was broadly characterized and designated as a candidate for the CLEC12A program AB0192.

Molecular format and design AB0192 is a heterodimeric trifunctional antibody that redirects NK and T cell activity to cancer cells expressing CLEC12A. AB0192 contains three chains: chain H, chain L, and chain S. A schematic diagram of the AB0192 molecule is shown in FIG. Chain H is the heavy chain of A49M-I that was chosen for its low affinity while being a potent agonist of the NKG2D receptor on NK cells and cytotoxic T cells. The M-I mutation was introduced to replace the Met102 residue in CDRH3, which is sensitive to oxidation and thus may exhibit liability. Chain L is the light chain of the anti-NKG2D targeting antibody A49M-I. Chain S contains an scFv that binds with high affinity to CLEC12A on AML cancer cells. Both the scFv and Fab domains are fused to human IgG1 Fc with native unmodified sequences in the CH2 domain and the antibodies bind to Fc receptors. Both chains have several mutations in the CH3 domain introduced to ensure stable heterodimerization of the two Fc monomers. There are 7 such mutations in the IgG1 Fc of AB0192: 3 mutations in chain H (K360E, K409W, Y349C) and 4 mutations in chain S (Q347R, D399V, F405T and S354C). Two of these mutations are involved in the formation of stabilized SS bridges (Y349C and S354C) between chain H and chain S (EU nomenclature). The scFv of chain S (VH-VL orientation) is stabilized by two mutations that introduce SS bridges ((H44)C and (L100)C, Chothia nomenclature). Chain S also contains a (G4S)4 non-immunogenic linker between VH and VL of the scFv. The amino acid sequence of the scFv targeting CLEC12A was derived from the humanized 9F11.Fv obtained from a hybridoma (Green Mountain Antibodies, Burlington, Vt.). Based on the sequence of the B7 antibody. The amino acid sequence of NKG2D Fab is based on the fully human antibody A49M-I obtained using human yeast display technology (Adimab, Lebanon, NH).

Description of heterodimerization mutations The EU numbering convention was used to annotate the amino acid residues of Fc.

Chain H contains the following mutations:
Heterodimerization mutations in the CH3 domain of the K360E-Fc region.
Heterodimerization mutations in the CH3 domain of the K409W-Fc region.
Y349C Intersubunit disulfide stabilizing mutation in the CH3 domain of the -Fc region.

The K360E/K409W mutations in the Fc region of chain H are designed to favor heterodimer interactions with chain S due to long range electrostatic interactions in addition to hydrophobic/conformational complementarity. (Choi et al., (2013) Mol Cancer Ther 12(12):2748-59; Choi et al., (2015) Mol Immunol 65(2):377-83). Mutation Y349C was introduced to create an additional interchain disulfide bond Y349C-(CH3-chain H)-S354C(CH3-chain S) pair (Choi et al., (2015) Mol Immunol 65(2) : 377-83.).

Chain S contains the following mutations:
Disulfide-stabilizing mutation in the VH region of G44C-scFv.
Disulfide stabilizing mutation in the VL region of Q100C-scFv.
Heterodimerization mutations in the CH3 domain of the Q347R-Fc region.
Heterodimerization mutations in the CH3 domain of the D399v-Fc region.
A heterodimerization mutation in the CH3 domain of the F405T-Fc region.
S354C - An intersubunit disulfide stabilizing mutation in the CH3 domain of the Fc region.

Mutations G(H44)C and Q(L100)C were introduced to stabilize the scFv by an engineered disulfide bond between VH and VL. Disulfide stabilization improves scFv structural and thermal stability. The Q347R/D399V/F405T mutations in the Fc region of chain S provide heterodimer-favorable interactions with chain H due to long-range electrostatic interactions in addition to hydrophobic/conformational complementarity (Choi et al, (2013) Mol Cancer Ther 12(12):2748-59). Mutation S354C was introduced to create an additional interchain CH3 disulfide bond with the Y349C (CH3-chain H)-S354C (CH3-chain S) pair (Choi et al., (2015) Mol Immunol 65 (2 ): 377-83).

Amino Acid Sequence The amino acid sequences of the three polypeptide chains that make up AB0192 are shown in FIG. A summary of the CDRs is shown in Table 204.

Analysis of potential sequence liability There is no predicted liability for chain L (NKG2D binding light chain). Chain H (NKG2D-bound heavy chain) has a DP in CDRH3. This motif is immune to forced decomposition. Therefore, this sequence was unchanged in AB0089. Chain S contains Met (potential oxidation site) and DG (potential isomerization site) in CDRH3. No preemptive ablation of M or DG of CDRH3 was attempted. Modifications of these residues were carefully monitored during accelerated stability and forced degradation studies. As shown below, no changes in Met or DG were observed at any of the stresses tested.

Framework Evaluation The CLEC12A binding arm of AB0192 was discovered by immunizing mice. The frameworks chosen for humanization are frequently used in clinical stage therapeutic monoclonal antibodies (Figure 211). The NKG2D binding arms are fully human, based on frameworks frequently used in advanced stage mAbs, and contain no aberrant residues or germline deviations.

Backmutations Molecular modeling was used to select mouse backmutations that help preserve the CDR architecture (Table 205). Seven backmutations were introduced: 3 in HC and 4 in LC.

Structural Modeling In the absence of crystal structures, structural models of the variable segments of the CLEC12A and NKG2D binding arms were generated for comparison. Molecular modeling was performed using the SAbPred website (opig.stats.ox.ac.uk/webapps/newsabdab/sabpred/).

Surface charge distribution and hydrophobic patch analysis of the CLEC12A binding arm of AB0192. ). The charge distribution of the anti-CLEC12A scFv is polarized ('top view', bottom panel), with negatively charged residues predominantly within CDRH3 and CDRL2. The uneven distribution of electrostatic patches on the paratope is likely target-related and may reflect the complementarity of the charge distribution on the cognate epitope, which is similar to that of CLEC12A and AB0192. may contribute to the high-affinity interactions between scFvs.

Figure 213 shows analysis of the CDR length and surface hydrophobic patches of the CLEC12A targeting arm of AB0192. The analysis is based on the therapeutic antibody profile (TAP): opig. stats. ox. ac. Done using uk/webapps/newsabdab/sabpred/tap. Candidates are compared with reference to 377 late stage therapeutic antibodies. The CDR lengths of the CLEC12A binding arm of AB0192 are within the typical range for late stage therapeutic antibodies. Hydrophobic patch analysis of the antibody's CDRs predicts its development potential. The theoretical hydrophobic character of the TAA arm of AB0192 is well within the criteria of commercialized biotherapeutics and late-stage therapeutic antibody candidates.

FIG. 214 shows the analysis of patches containing positive and negative charges and the symmetry of these charges around the CDR regions. All three charts show that the charge distribution is within the norm compared to advanced stage therapeutic antibodies.

Surface charge distribution and hydrophobic patch analysis of NKG2D binding arms of AB0192 NKG2D binding Fabs are shown in ribbon diagrams with three different orientations (Fig. 215, top panel) and the corresponding surface charge distributions at the same orientations are provided. (Fig. 215, bottom panel). The surface charge distribution of the NKG2D A49M-I arm is more evenly distributed than that of the CLEC12A targeting arm of the molecule.

FIG. 216 shows patches of CDR length and surface hydrophobicity analysis of the NKG2D targeting arm of AB0192. The CDR length, hydrophobicity, positive/negative charge distribution of the NKG2D targeting arm of AB0192 appear to be well within the criteria of existing therapeutic antibodies.

FIG. 217 shows the analysis of patches containing positive and negative charges and the symmetry of these charges around the CDR regions. All three charts show that the charge distribution is within the norm compared to the therapeutic antibody population.

Prediction of Immunogenicity The protein sequences of the three chains of AB0192 were examined for the presence of potentially immunogenic T-cell epitopes using the EpiMatrix algorithm and ranked for immunogenic potential (FIG. 218). ). AB0192 scores favorably on the Treg Modulated Immunogenic Protein Scale. The Treg-adjusted Epimatrix protein scores for the three chain sequences of AB0192 are chain H: -33.39, chain S: -19.94 and chain L: -23.49 (Table 206). The predicted risk of immunogenicity of AB0192 appears to be low.

Expression Expression of AB0192 was achieved using the Dragonfly Tx F3'TriNKET platform purification method. Briefly, AB0192 was transformed into Expi293 and Expi293 by three plasmids encoding an NKG2D-targeting light chain (chain L), an NKG2D-targeting IgG heavy chain (chain H), and a CLEC12A-targeting scFv-Fc fusion (chain S). It was expressed by transient transfection of ExpiCHO cells. For expression of both Expi293 and ExpiCHO, chain S:chain H:chain L was transfected at a plasmid ratio of 4:1:1 to maximize the proportion of the desired heterodimeric product. This ratio was obtained empirically based on previously developed humanized TriNKETs and was not specifically optimized for AB0192.

Purification Purification of AB0192 was performed using a two-step F3'TriNKET platform purification method. No optimization or extensive process development for AB0192 purification was performed. A schematic of the ExpiCHO expressed AB0192 purification (lot AB0192-005) is shown in FIG.

Two lots of AB0192 were produced in transiently transfected ExpiCHO and Expi293 cells. Table 207 summarizes the titer and final product purity for both lots.

The identity of AB0192 was confirmed by mass spectrometry. LC-MS analysis of intact AB0192 (lot AB0192-005) showed an observed mass (126,429.9 Da) (Figure 220) in good agreement with the theoretical mass of 126,429.5 Da. The predominant glycosylation pattern observed (G0F/G0F) is typical for Fc-containing molecules expressed in CHO cells.

Biochemical and Biophysical Properties AB0192 was characterized by SEC, CE, cIEF, HIC, and DSC and confirmed to have the appropriate purity and predevelopment properties of a superior biotherapeutic. rice field.

Purity The purity of AB0192 (Lot AB0192-005) as assessed by SEC, non-reduced and reduced CE-SDS is summarized in Table 208.

Size Exclusion Chromatography SEC analysis of purified AB0192 (lot AB0192-005) showed that the integrated peak areas confirmed greater than 99% monomer (Figure 221).

Non-reducing and reducing CE-SDS
AB0192 is highly pure by CE-SDS (Figure 222). Under non-reducing conditions (left panel), the major impurities are induced by the method (free light chain and TriNKET-LC). Under reducing conditions (right panel) three predicted chains (light (L), heavy (H) and scFv-Fc chains (S)) are observed.

Experimental pI and charge profile by cIEF The cIEF profile of AB0192 (Lot AB0192-005) is shown in (Figure 223). AB0192 has a major peak at a pI of 8.9±0.0. Some smaller, overlapping acidic and minor basic peaks are also observed.

Hydrophobicity Assessed by HIC The hydrophobicity of AB0192 was assessed using hydrophobic interaction chromatography (HIC) (Figure 224 and Table 209). The chromatogram shows a single narrow peak, indicating that the molecule is very pure and homogeneous. The retention time of AB0192 is comparable to that of Humira, a therapeutic monoclonal antibody that functions normally.

Thermal stability by DSC The thermal stability of AB0192 was evaluated by DSC in several buffers (PBS pH 7.4, HST pH 6.0, CST pH 7.0). AB0192 showed high thermal stability in all buffers tested (Figure 225, Table 210). Tm1 corresponding to scFv development met the criteria of 65° C. or higher assumed in thermostability evaluation.

Assignment of Disulfide Bonds AB0192 is an engineered molecule based on the backbone of a monoclonal IgG1 antibody. A typical IgG1 contains 16 disulfide bonds, whereas the F3' format of AB0192 has only 15 disulfide bonds. Half of AB0192 is an anti-NKG2D half antibody and thus contains the expected seven disulfide bonds (one in each IgG domain and an intermolecular disulfide connecting LC to HC). The other half of AB0192 is an anti-CLEC12A scFv-Fc fusion. The scFv contains three disulfide bonds (one in each anti-CLEC12A VH and VL domain and an engineered stabilizing disulfide bridging the variable domains) and the Fc contains two predicted disulfides in the IgG domain. . The heterodimer is held together with two hinge disulfides and one additional intermolecular engineering disulfide connecting Fab-Fc to scFv-Fc in the CH3 domain. Collectively, this results in 15 predicted disulfide bonds in AB0192. A predicted disulfide map of AB0192 is shown in FIG.

Disulfide bond connectivity of AB0192 was confirmed by LC-MS/MS peptide mapping analysis of non-reduced digests. Disulfide-linked peptides were identified by MS/MS database searches and confirmed by comparing intensities in native and reduced digestions. To observe the engineered scFv-stabilizing disulfides, AB0192 was digested with both trypsin and chymotrypsin. Figure 227 shows extracted ion chromatograms (XIC) of engineered disulfide pairs of scFv (non-reduced and reduced) and the strongest charge states of the peptide pairs. All other disulfides were identified after digestion with trypsin alone. The XIC in Figure 228 confirms the presence of engineered SS bridges introduced to stabilize Fc heterodimerization. A summary of disulfide-linked peptides observed in AB0192 is shown in Table 211. All disulfide-linked peptides observed were observed with high mass accuracy (less than 5 ppm), were reducible, and sequence confirmed by MS/MS fragmentation.

Binding Characterization Binding to Human CLEC12A The affinity of AB0089 for human CLEC12A was determined by SPR at 37°C. AB0089 binds to human CLEC12A with high affinity (Figure 229, Table 212). According to the TriNKET MOA, the molecule is designed to bind strongly to TAA and weakly to NKG2D and CD16a. The complex between AB0192 and human CLEC12A is potent as evidenced by a slow dissociation constant of 1.40±0.09×10 ⁻³ s ⁻¹ . The equilibrium binding affinity _KD of AB0192 is 1.81±0.16 nM.

Binding to Polymorphic Variants of Human CLEC12A (Q244)
The polymorphic variant CLEC12A (Q244) is prevalent in 30% of the population. We tested the binding of AB0192 to this genetic variant by SPR and compared its affinity to CLEC12A(K244) WT (Figure 230). The binding affinity of AB0192 to the WT and (K244Q) variants of CLEC12A is within 2.5-fold (Table 213). Binding of AB0192 to cynomolgus monkey CLEC12A (cCLEC12A) was also tested. No quantifiable binding was observed at a concentration of 100 nM.

Binding to Differentially Glycosylated CLEC12A Human CLEC12A is a highly glycosylated protein (6 potential N-glycosylation sites with an ECD that compromises 201 amino acids). Variation in the glycosylation status of CLEC12A on the surface of different cell types has been described in the literature (Marshall et al., (2006) Eur. J. Immunol., 36:2159-2169). CLEC12A expressed on the surface of AML cells from different patients may have different glycosylation patterns as well. To confirm that AB0089 binds to different glycovariants of human CLEC12A, the antigen was treated with neuraminidase (to remove terminal sialic acid) or PNGaseF (to remove N-linked glycans) to Affinity was compared to untreated CLEC12A by SPR. AB0192 is expected to bind both glycosylated and deglycosylated variants of CLEC12A (Figure 231 and Table 214), insensitive to heterogeneity in target glycan composition.

Binding to CLEC12A+ Isogenic and Cancer Cell Lines Assessment of AB0192 binding to both isogenic cell lines expressing CLEC12A and AML cancer cells was monitored by FACS (Figure 232, and Table 215). AB0192 exhibits high affinity for cell surface expressed CLEC12A on isogenic Ba/F3-CLEC12A cells and binds AML cell lines PL-21 and HL-60 with an EC50 of less than 1 nM. The specificity of AB0089 for CLEC12A was demonstrated by the lack of binding to parental Ba/F3 cells that do not express the target.

Binding to Human NKG2D Binding of AB0192 to human NKG2DECD by SPR at 37° C. (Biacore) (FIG. 233). Recombinant mFc-tagged NKG2D dimers were used for this experiment, as NKG2D is dimeric in nature. Affinity was determined using AB0192. Figure 233 shows a binding sensorgram of AB0192 to human NKG2D. To obtain equilibrium affinity data, two different fits were utilized: a steady-state affinity fit and a kinetic fit. Kinetic constants and equilibrium affinity constants are shown in Table 216. AB0192 was designed to bind human NKG2D with low affinity and, most importantly, a fast dissociation rate. The dissociation rate constant was (1.46±0.06×10−1 s−1). Equilibrium affinity constants (KD) obtained by kinetic and steady-state affinity fits are very similar, 779±10 nM and 781±9 nM, respectively, indicating high confidence in the parameters measured. It suggests.

One mode of AB0192 mode of action binding to human CD16a (V158 allele) is through human CD16a engagement (FCγRIIIa) expressed on NK cells via its Fc. Binding of AB0192 to human CD16a was tested by SPR (Figure 234, Table 217). The CD16a high affinity V158 allele was used for this experiment. Trastuzumab (Herceptin) was included as a human IgG1 control because, according to the literature, Fc receptor affinity values for IgG1 vary with experimental design and receptor procurement. The affinity of AB0192 for both alleles is comparable to that of trastuzumab for the same receptor (approximately 2-fold difference). We do not expect this difference to have any meaningful physiological significance.

CD16a-NKG2D synergy study AB0192 is a trifunctional IgG-based biologic with two modes of binding to NK cells. CD16a binding is achieved through Fc and NKG2D binding through A49M-IFab. Both interactions are important for optimal cytotoxic activity of AB0192. To demonstrate the synergistic effect of co-engagement of human CD16a and human NKG2D binding, we performed SPR experiments to qualitatively compare binding of AB0192 to NKG2D, CD16a, and mixed NKG2D-CD16a Biacore chip surfaces. gone. Although the affinity of AB0192 for human NKG2D and human CD16a are both low, binding to both targets simultaneously produces an affinity effect manifested as a slower off-rate (Figure 235). Therefore, AB0192 may strongly engage CD16a and NKG2D.

Co-engagement of CLEC12A and NKG2D To determine whether binding of one target interferes with binding of the other target to AB0192, CLEC12A and NKG2D were co-engaged on AB0192 captured on an anti-hFc IgG SPR chip. Infused continuously (Figure 236). Target binding sensorgrams show that CLEC12A occupancy does not interfere with NKG2D binding (Figure 236, upper panel) and vice versa (Figure 236, lower panel). Kinetic parameters are not significantly affected by target occupancy of the AB0192 molecule due to the similarity in shape of the respective sensorgram segments showing binding of each target to free AB0192 and AB0192 saturated with other targets. is suggested. For example, the shape of the CLEC12A binding segment in the sensorgrams is similar in both panels. Furthermore, the lack of effect on the relative stoichiometry of each target binding (when compared to binding to unoccupied AB0192) suggests that the NKG2D and CLEC12A binding sites on AB0192 are completely independent. (Table 218). Thus, AB0192 successfully achieves simultaneous co-engagement of the tumor antigen and the NKG2D targeting arm.

Binding to Fcγ Receptors As AB0192 is an IgG1-derived biodrug candidate, it is important to maintain proper Fc receptor binding properties. SPR data suggest that AB0089 binds to human and cynomolgus monkey Fcγ receptors with affinity comparable to trastuzumab, a commercial IgG1 biologic that serves as an experimental control (Table 219). A detailed description of each individual receptor binding is provided below.

Binding to Human and Cynomolgus Monkey CD64 (FcγRI)
Table 220 shows that AB0089 binds to recombinant human and cynomolgus CD64 (FcγRI) with affinities comparable to trastuzumab (less than 2-fold difference). Figures 237 and 238 show the binding of AB0089 to human and cynomolgus monkey CD64, respectively.

Binding to human CD32a (FcγRIIa)
There is no meaningful difference between the affinities of both alleles of human CD32a for AB0089 and trastuzumab (Table 221). Figures 239 and 240 show binding of AB0089 and trastuzumab, respectively, to CD32a (H131 and R131 alleles) as tested by SPR.

Binding to Human CD16aF158 (FcγRIIIa F158) and Cynomolgus Monkey (cyno) CD16 A detailed description of AB0192 binding CD16a V158 is provided in Section 6.9.6. The binding of AB0192 to the low affinity F158 allele of human FcγRIIIa and cynomolgus monkey FcγRIII is shown in Table 222, Figures 241 and 242. AB0192 binding affinity values for recombinant human CD16a (F158 allele) and cynomolgus monkey CD16 are comparable to those for trastuzumab, with less than a 2-fold difference in affinity (Table 222).

Binding to human CD32b (FcγRIIb)
There is no meaningful difference between the affinity of CD32b for AB0089 and trastuzumab (Table 223). Figure 243 shows binding of AB0089 and trastuzumab to CD32b as examined by SPR.

Binding to human CD16b (FcγRIIb)
AB0089 binds human CD16b (FcγR3b) with affinity (less than 2-fold difference) comparable to one trastuzumab (Table 224). Figure 244 shows binding of AB0089 and trastuzumab to CD16b as examined by SPR.

Binding to Human and Cynomolgus FcRn Figures 245 and 246 show the binding of AB0192 to human and cynomolgus monkey FcRn, respectively, as tested by SPR. The affinity of AB0192 for human and cynomolgus FcRn is similar to that of the commercial IgG1 biologic, trastuzumab, which served as an experimental control (Table 225).

Epitope binning We compared the binding epitopes of AB0237, AB0089, and AB0192 associated with three reference control TriNKETs by SPR (Figure 247). cFAE-A49. Tepositamab is based on the CLEC12A binding arm of Merus' tepositamab (WO_2014_051433_A1), cFAE-A49. CLL1-Merus is based on Merus clone 4331 (WO_2014_051433_A1) and cFAE-A49. h6E7 is based on the h6E7 mAb from Genentech (h6E7) (US_2016_0075787_A1) The interaction of AB0192 with hCLEC12A was blocked by all three control molecules cFAE-A49. Tepositamab, cFAE. A49. h6E7, and cFAE-A49. CLL1-Merus suggests that the AB0192 binding epitope is broad and overlapping with both the Merus and Genentech molecules. This footprint differs from AB0237 and AB0089, which cross-block only with Merus molecules. Therefore, the binding epitope of AB0192 is different from AB0089 and AB0237.

Efficacy The ability of AB0192 to lyse human cancer cells expressing CLEC12A was determined in a human NK cell-mediated cytotoxicity assay. AB0192 showed high potency in killing PL21 AML cells, significantly exceeding the corresponding monoclonal antibodies (Figure 248 and Table 226).

Specificity Non-Specific Binding Assessed by PSR Non-specific binding of AB0192 was assessed by a flow cytometry-based PSR assay measuring binding to preparations of detergent-solubilized CHO cell membrane proteins. (Fig. 249). The PSR assay correlates well with cross-interaction chromatography, which is a surrogate for antibody solubility, and baculovirus particle enzyme-linked immunosorbent assay, which is a surrogate for in vivo clearance, and thus serves as a good predictor of development potential. It is known to AB0192 showed no non-specific binding similar to the low PSR control (trastuzumab), suggesting high specificity and lack of non-specific binding to irrelevant proteins.

Specificity Evaluation of AB0192 in HuProt™ Microarray Assay To investigate the specificity of AB0192, we investigated protein array technology. The HuProt™ Human Proteome Microarray provides the largest database of individually purified full-length human proteins on a single microscope slide. An array of 22,000 full-length human proteins was expressed in the yeast Cerevisiae, purified, and then printed in duplicate on microarray glass slides, allowing profiling of thousands of interactions in a high-throughput fashion. be. Figure 250 shows the relative binding (Z-score) of AB0192 at 1 μg/ml to human CLEC12A compared to the entire human proteome microarray. A Z-score is the average binding score of two replicates of a given protein. For comparison purposes, the top 24 proteins with remaining background binding to AB0192 are also shown in FIG. Table 227 shows the Z-score and S-score of AB0192 against human CLEC12A and the top 6 proteins on the microarray. The S-score is the difference between the Z-score of a given protein and one rank next to it. Based on Z and S score criteria, AB0192 showed high specificity to humans and lack of off-target binding in the HuProt™ Human Proteome Assay.

Development feasibility evaluation The stability and forced degradation of AB0192 were evaluated under the following conditions.

Accelerated (40° C.) Stability—20 mg/ml in 20 mM histidine, 250 mM sucrose, 0.01% Tween®-80 (pH 6.0), 40° C., 4 weeks (SEC, SPR, potency, SDS- CE)
- 4 weeks at 40°C in 20 mM sodium citrate, 250 mM sucrose, 0.01% Tween®-80 (pH 7.0) (SEC, SPR, potency, SDS-CE)

Chemical stability - oxidation: 0.02% hydrogen peroxide, 1 mg/ml in PBS (room temperature), 24 hours (SEC, CE-SDS, SPR, potency, peptide map)
- pH 8.0: 1 mg/ml 20 mM Tris, 40°C, 2 weeks (cIEF, SPR, potency, peptide map)
- pH 5.0: 1 mg/ml 20 mM sodium acetate, 40°C, 2 weeks (cIEF, SPR, potency, peptide map).

Manufacturability - Freeze/Thaw: 20 mg/ml 6 cycles of 20 mM sodium citrate, 250 mM sucrose, 0.01% Tween®-80, pH 7.0 (SEC, SDS-CE)
- Agitation: 20 mg/ml (SEC, SDS-CE) in 20 mM sodium citrate, 250 mM sucrose, 0.01% Tween®-80 (pH 7.0)
- High concentration: >150 mg/ml, PBS (SEC, A280)
- Low pH hold: pH 3.3, 1.0 h, ProA elution buffer (SEC, cIEF, SDS-CE, intact mass, SPR, potency)

Accelerated (40° C.) Stability To assess the stability of AB0192, the molecule was treated with (1) 20 mg/ml in 20 mM histidine, 250 mM sucrose, 0.01% PS-80, pH 6.0 (HST), and (2) Staged at 40° C. for 4 weeks under two different buffer conditions of 20 mg/ml in 20 mM citrate, 250 mM sucrose, 0.01% PS-80, pH 7.0 (CST). Stability of both buffers was evaluated by SEC, CE, SPR and potency.

Stability in HST, pH 6.0 Minimal aggregation or fragmentation by SEC (Figure 251, Table 228) or CE-SDS (Figure 252, Table 229) after 4 weeks at AB0192 in HST, pH 6.0 at 40°C showed transformation. Monomer loss was 1.6% by SEC and 1% by non-reduced CE. There is no significant difference in active species content or affinity of hCLEC12A, hNKG2D, and hCD16a between control and stress samples (Figure 253 and Table 230). Finally, there was no difference in potency between control and stress samples tested in the KHYG-1-CD16aV cytotoxicity assay (Figure 254 and Table 231).

Stability at CST pH 7.0 After 4 weeks at 40° C., AB0192 at CST, pH 7.0 showed minimal aggregation (FIG. 255 and Table 232) and fragmentation (FIG. 256 and Table 233). A 1.6% and 0.8% loss of monomer was observed by SEC and non-reducing CE-SDS, respectively. There is no significant difference in active species content or affinity of hCLEC12A, hNKG2D, and hCD16a between control and stress samples (Figure 257 and Table 234). Finally, there was no difference in potency between control and stress samples in the KHYG-1-CD16aV cytotoxicity assay (Figure 258 and Table 235).

Chemical Stability Forced Oxidation After 24 hours of oxidative stress in 0.02% hydrogen peroxide, AB0192 showed no significant decrease in monomer content (Figure 259 and Table 236). The purity assessed by CE remained high (Figure 260 and Table 237). The amount and affinity of active species for CLEC12A, NKG2D, and CD16aV were unchanged (Figure 261 and Table 238) and potency was similar to control samples (Figure 262 and Table 239).

Site-specific oxidation of methionine was monitored after forced oxidation by tryptic peptide mapping. As expected for IgG1, as expected (Table 240), significant levels of oxidation were detected only at the two methionines of Fc. Our historical data show that the same residues are modified in 61.6% and 39.1% of trastuzumab under the same conditions.

Oxidation of tryptophan residues in the AB0192 CDR was also monitored in the same experiment. None of the tryptophan residues had significantly increased oxidation after stress (Table 241).

pH 5 Stress The chemical stability of AB0192 was evaluated at low pH (20 mM sodium acetate, pH 5.0, 40° C., 2 weeks). No monomer loss (Figure 263 and Table 242) was observed by SEC after 2 weeks at pH 5. The difference in purity detected by non-reduced and reduced CE-SDS was less than 1% (Figure 264 and Table 243). There were no significant differences in CLEC12A, NKG2D, or CD16aV affinity of the amount of active species observed (Figure 265 and Table 244). A small acidic shift in the overall charge profile was detected by cIEF (Figure 266 and Table 245), but this acidic shift did not affect the potency of AB0192 (Figure 267 and Table 246).

ｐＨ５及びｐＨ８のストレスを受けた物質のペプチドマッピング分析 pH 8 Stress The chemical stability of AB0192 was also evaluated by incubation at high pH (20 mM Tris, pH 8.0, 40° C., 2 weeks). After 2 weeks at pH 8, there was a 1.6% loss of monomer by SEC (Figure 268 and Table 247). Slight differences in purity were detected by non-reduced and reduced CE-SDS (Figure 269 and Table 248). There were no significant differences in the affinities or amounts of the active species CLEC12A, NKG2D, or CD16aV observed (Figure 270 and Table 249). A significant acidic shift in the overall charge profile detected by cIEF was detected (Figure 271 and Table 250), which could be attributed to deamidation of the entire molecule. However, this acidic shift did not significantly affect the potency of AB0192 (Figure 272 and Table 251).

Peptide mapping analysis of pH 5 and pH 8 stressed substances

To determine whether the pH stress-induced changes observed by cIEF are due to complementarity determining region (CDR) modifications (alterations), the same pH 5 and pH 8 stress samples were analyzed by LC-MS/MS. Analyzed by peptide map. Based on peptide map data, all CDRs of AB0192 are resistant to modification during low and high pH stress (Table 252). No evidence of aspartic isomerization was observed based on manual inspection of peak shape.

Manufacturability Freeze/Thaw Stability An aliquot of AB0192 (20 mg/ml in CST pH 7.0) was placed in a styrofoam container (to slow down the freezing rate) and the container placed at -80°C. Freeze and thaw six times. Once frozen, the container was placed on the lab bench and an aliquot was allowed to thaw. After 6 cycles of freeze/thaw, AB0192 showed no loss of monomer by SEC and protein concentration remained the same (Figure 273 and Table 253).

攪拌 No loss of purity was detected by reducing or non-reducing CE-SDS after 6 freeze/thaw cycles (Figure 274 and Table 254).

stirring

Agitation studies were performed in CST, pH 7.0 buffer. AB0192 was incubated for 24 hours with constant steering (60 rpm) in a glass vial equipped with a micro stir bar. No loss of monomer was detected by SEC compared to control samples (Figure 275 and Table 255).

There was no increase in fragmentation detected by reduced or non-reduced CE-SDS after 24 hours of agitation (Figure 276 and Table 256).

Low pH Hold A low pH hold study was performed to determine if AB0192 could be amended to the commonly used low pH hold protocol used for virus inactivation in biologics manufacturing. Incubation at low pH can compromise the biophysical stability and integrity of the molecule and can cause chemical degradation of amino acid side chains, especially isomerization of aspartic acid. CDRH3 of chain S has potential sequence liability DS. Therefore, we applied a wide range of assays to assess protein quality after low pH holding. Most of the AB0192 protein A eluate was immediately adjusted to neutral pH and a small sample was adjusted to pH 3.7 and held at room temperature for 1 hour to mimic a typical viral inactivation step. After the holding period, the protein A eluate was neutralized with 1.0 M Tris, pH 8.3 to achieve neutral pH. Analytical size exclusion chromatography was performed to determine if there was a change in profile or aggregate content before and after low pH exposure (Figure 277). No change in profile was observed after the low pH hold. Both aliquots were further purified by a second ion exchange chromatography and subjected to a panel of characterization assays.

Chemical modifications of amino acid side chains can usually be observed globally using cIEF. The cIEF profiles of the AB0192 control and low pH hold lots are visually very similar, with relative quantification of acidic, major, and basic species all within 2% of each other. It shows that low pH retention had no measurable effect on the charge profile of AB0192 (Figure 278 and Table 257).

Affinities for hCLEC12a, hNKG2D, and CD16aV did not change after the low pH holding step (Figure 279 and Table 258). This indicates that AB0192 retains affinity for all three targets under low pH holding conditions used as a virus inactivation step in biologics manufacturing.

The potency of the low pH retention samples was further tested against CLEC12A+PL-21 cancer cells in the KHYG1-CD16aV cytotoxicity bioassay. The potency of AB0192 did not change after the low pH holding step (Figure 280 and Table 259).

Collectively, these results demonstrate that the molecular format and structure of AB0192 fulfills the conceptual requirements for new multispecific therapeutics that simultaneously engage CLEC12A, NKG2D, and CD16a to treat AML. be done. AB0192 is potent and has a favorable developability profile, with neither sequence liability nor significant post-translational modifications observed under any stress. AB0192 binds to a different epitope than AB0237 and AB0089 TriNKET.

INCORPORATION BY REFERENCE Unless stated to the contrary, the entire disclosure of each of the patent documents and scientific articles referenced herein is incorporated by reference for all purposes.

Equivalents The application may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Accordingly, the foregoing embodiments are to be considered in all respects illustrative rather than limiting of the application described herein. The scope of the present application is, therefore, indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein. be done.
arrangement

In some embodiments, ALL is selected from B-cell acute lymphoblastic leukemia (B-ALL) and T-cell acute lymphoblastic leukemia (T-ALL). In some embodiments, the MPN is selected from polycythemia vera, essential thrombocythemia (ET), and myelofibrosis. In some embodiments, the non-Hodgkin's lymphoma is selected from B-cell lymphoma and T-cell lymphoma. In some embodiments, the lymphoma is chronic lymphocytic leukemia (CLL), lymphoblastic lymphoma (LPL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), mediastinal primary Cellular B-cell lymphoma (PMBL), follicular lymphoma, mantle cell lymphoma, pilocytic leukemia, plasma cell myeloma (PCM) or multiple myeloma (MM), mature T/NK cell tumors, and histiocytic tumors is selected from In certain embodiments, the cancer expresses CLEC12A.
In certain embodiments, for example, the following are provided:
(Item 1)
is a protein and:
(a) a first antigen binding site that binds to NKG2D;
(b) a second antigen binding site that binds CLEC12A; and
(c) comprising an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen binding site that binds CD16;
wherein said second antigen-binding site that binds to CLEC12A is:
(i) a heavy chain comprising complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3), each comprising the amino acid sequences of SEQ ID NOS: 137, 272, and 273; a variable domain (VH); and a light chain variable domain (VL) comprising CDR1, CDR2 and CDR3, each comprising the amino acid sequences of SEQ ID NOs: 140, 141 and 142;
(ii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139 respectively; VL containing;
(iii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOS: 251, 196 and 246 respectively; VL containing;
(iv) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:221, 166 and 223 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:240, 226 and 179 respectively VL containing;
(v) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:206, 166 and 241 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:245, 239 and 179 respectively VL containing;
(vi) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:221, 236 and 223 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:152, 226 and 179 respectively; VL containing;
(vii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 159, 170 and 183 respectively; VL containing;
(viii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 197, 202 and 207 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 211, 212 and 214 respectively VL;
(ix) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:220, 222 and 257 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:224, 225 and 227 respectively; VL containing; or
(x) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:230, 231 and 232 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:233, 234 and 235 respectively; VL, including
(Item 2)
The protein of item 1, wherein said second antigen binding site that binds to CLEC12A comprises
(i) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 272, and 273, respectively; VL; or
(ii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139 respectively; including VL,
The protein, comprising:
(Item 3)
The protein of item 1 or 2, wherein
(i) said VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:335, and said VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:336;
(ii) said VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:270, and said VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:271; or
(iii) said protein, wherein said VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:178, and said VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:289;
(Item 4)
The protein according to any one of items 1 to 3,
(i) said VH comprises the amino acid sequence of SEQ ID NO:335 and said VL comprises the amino acid sequence of SEQ ID NO:336;
(ii) said VH comprises the amino acid sequence of SEQ ID NO:270 and said VL comprises the amino acid sequence of SEQ ID NO:271;
(iii) said protein wherein said VH comprises the amino acid sequence of SEQ ID NO:178 and said VL comprises the amino acid sequence of SEQ ID NO:289;
(Item 5)
143 and 144; 178 and 144; 256 and 144; 143 and 163; 143 and 167; 143 and 171; , item 1 or 2.
(Item 6)
Said second antigen binding site is present as a single chain fragment variable (scFv), wherein said scFv comprises 6. The protein of any one of items 1-5, comprising an amino acid sequence selected from 161, 164, 165, 168, 169, 172, 173, 176, 177, 180 and 181.
(Item 7)
7. The protein of item 6, wherein said scFv comprises the amino acid sequence of SEQ ID NO:274 or item 180.
(Item 8)
a VH wherein said second antigen-binding site that binds CLEC12A comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 251, 196, and 246, respectively; and amino acids of SEQ ID NOs: 198, 199, and 201, respectively. 2. The protein of item 1, comprising a VL comprising CDR1, CDR2, and CDR3 comprising sequences.
(Item 9)
wherein said second antigen binding site that binds to CLEC12A is:
(i) VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 195, 196, 187 respectively; VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 198, 199, 201 respectively; ;
(ii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 192, 196 and 187 respectively; VL containing; or
(iii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOS: 192, 196 and 213 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOS: 198, 199 and 201 respectively; VL including
The protein of item 1, comprising:
(Item 10)
any one of items 1 and 8-9, wherein said VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 148, and said VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 203 Proteins as described in.
(Item 11)
11. The protein of any one of items 1 and 8-10, wherein said VH comprises the amino acid sequence of SEQ ID NO:148 and said VL comprises the amino acid sequence of SEQ ID NO:203.
(Item 12)
162 and 218; 148 and 218; or 210 and 218.
(Item 13)
said second antigen binding site is present as a scFv, wherein said scFv is selected from SEQ ID NOs: 184, 185, 204, 205, 208, 209, 215, 216, 252, 253, 254, and 255 13. The protein according to any one of items 8 to 12, comprising an amino acid sequence of
(Item 14)
14. The protein of item 13, wherein said scFv comprises the amino acid sequence of SEQ ID NO:204.
(Item 15)
3. A protein according to any one of the preceding items, wherein said second antigen binding site binds to CLEC12A in a glycosylation-independent manner.
(Item 16)
is a protein and:
(a) a first antigen binding site that binds to NKG2D;
(b) a second antigen binding site that binds CLEC12A; and
(c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen binding site that binds CD16;
wherein said second antigen binding site binds to CLEC12A in a glycosylation-independent manner.
(Item 17)
17. The protein of any one of items 1-16, wherein said second antigen binding site binds to human CLEC12A with a dissociation constant (K _D ) of 20 nM or less, as measured by surface plasmon resonance (SPR).
(Item 18)
18. The protein of any one of items 1-7 and 16-17, wherein said second antigen binding site binds human CLEC12A with a K _D of 1 nM or less as measured by SPR.
(Item 19)
19. The protein of any one of items 1-7 and 15-18, wherein said second antigen binding site binds to human CLEC12A comprising a K244Q substitution.
(Item 20)
20. The protein of any of items 1-19, wherein said protein comprises an antibody Fc domain, or a portion thereof sufficient to bind CD16.
(Item 21)
21. The protein of any one of items 1-20, wherein said first antigen binding site that binds NKG2D is a Fab fragment and said second antigen binding site that binds CLEC12A is an scFv.
(Item 22)
Any one of items 1-5, 8-12 and 15-20, wherein said first antigen binding site that binds NKG2D is a scFv and said second antigen binding site that binds CLEC12A is a Fab fragment. Proteins as described in.
(Item 23)
21. The protein of any one of items 1-20, further comprising an additional antigen binding site that binds to CLEC12A.
(Item 24)
24. The protein of item 23, wherein said first antigen binding site that binds NKG2D is a scFv and said second and said additional antigen binding site that binds CLEC12A are each a Fab fragment.
(Item 25)
24. The protein of item 23, wherein said first antigen binding site that binds NKG2D is a scFv, and said second and said additional antigen binding site that binds CLEC12A are each a scFv.
(Item 26)
26. The protein of any one of items 23-25, wherein the amino acid sequences of said second and said additional antigen binding sites are identical.
(Item 27)
is a protein and:
(a) a first antigen binding site that binds to NKG2D;
(b) a second antigen binding site that binds CLEC12A; and
(c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen binding site that binds CD16;
wherein said first antigen binding site that binds NKG2D is a Fab fragment and said second antigen binding site that binds CLEC12A is a scFv.
(Item 28)
of items 22, 24-26, wherein said scFv that binds NKG2D is linked via a hinge containing Ala-Ser or Gly-Ser to an antibody constant domain or part thereof sufficient to bind CD16 A protein according to any one of claims 1 to 3.
(Item 29)
Each scFv that binds CLEC12A is linked via a hinge containing an Ala-Ser or Gly-Ser to an antibody constant domain or part thereof sufficient to bind CD16, items 21, and 25- 29. The protein of any one of 28.
(Item 30)
30. A protein according to items 28 or 29, wherein said hinge further comprises the amino acid sequence Thr-Lys-Gly.
(Item 31)
any one of items 22, 24-26, 28, and 30, wherein within said scFv that binds to NKG2D, said scFv heavy chain variable domain forms a disulfide bridge with said scFv light chain variable domain. protein.
(Item 32)
any one of items 21, 25-27, and 30-31, wherein within each scFv that binds CLEC12A, said heavy chain variable domain of said scFv forms a disulfide bridge with said light chain variable domain of said scFv The protein according to the paragraph.
(Item 33)
33. The protein of item 31 or 32, wherein said disulfide bridge is formed between C44 of said heavy chain variable domain and C100 of said light chain variable domain, numbered under the Kabat numbering scheme.
(Item 34)
Any one of items 22, 24-26, 28, 30-31, and 33, wherein within said scFv that binds NKG2D, said heavy chain variable domain is linked to said light chain variable domain via a flexible linker. Proteins as described in.
(Item 35)
Any of items 21, 25-27, 29-30, and 32-34, wherein within each scFv that binds CLEC12A, said heavy chain variable domain is linked to said light chain variable domain via a flexible linker. 1. Protein according to item 1.
(Item 36)
36. The protein of item 34 or 35 , wherein said flexible linker comprises ( _G4S ) ₄ .
(Item 37)
any one of items 22, 24-26, 28, 30-31, 33-34, and 36, wherein in said scFv that binds NKG2D said heavy chain variable domain is located C-terminal to said light chain variable domain The protein according to the paragraph.
(Item 38)
any of items 21, 25-27, 29-30, 32-33, and 35-37, wherein within each scFv that binds CLEC12A, said heavy chain variable domain is located C-terminal to said light chain variable domain 1. Protein according to item 1.
(Item 39)
any one of items 22, 24-26, 28, 30-31, 33-34, and 36, wherein in said scFv that binds NKG2D said heavy chain variable domain is located N-terminal to said light chain variable domain The protein according to the paragraph.
(Item 40)
of items 21, 25-27, 29-30, 32-33, 35-36, and 39, wherein said heavy chain variable domain is located N-terminal to said light chain variable domain in each scFv that binds CLEC12A. A protein according to any one of claims 1 to 3.
(Item 41)
any one of items 21, 27, 29-30, 32-33, 35-36, 38 and 40, wherein the Fab fragment that binds NKG2D is not positioned between the antigen binding site and said Fc or portion thereof The protein according to the paragraph.
(Item 42)
of items 22, 24, 26, 28, 30-31, 33-34, 36-37, and 39, wherein the Fab fragment that binds CLEC12A is not positioned between the antigen binding site and said Fc or portion thereof A protein according to any one of claims 1 to 3.
(Item 43)
a VH wherein said first antigen-binding site that binds NKG2D comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:81, 82, and 112, respectively; and amino acids of SEQ ID NOs:86, 77, and 87, respectively. 43. A protein according to any one of items 1 to 42, comprising a VL comprising CDR1, CDR2 and CDR3 comprising sequences.
(Item 44)
The first antigen-binding site that binds to NKG2D is
(i) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS:81, 82, and 97, respectively; VL containing; or
(ii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:81, 82 and 84 respectively; and CDR1, CDR2 and CDR2 comprising the amino acid sequences of SEQ ID NOs:86, 77 and 87 respectively; VLs containing CDR3,
43. The protein of any one of items 1-42, comprising
(Item 45)
said VH of said first antigen binding site comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:95, and said VL of said first antigen binding site is an amino acid that is at least 90% identical to SEQ ID NO:85 45. A protein according to any of items 43 or 44, comprising a sequence.
(Item 46)
46. Any of items 43-45, wherein said VH of said first antigen binding site comprises said amino acid sequence of SEQ ID NO:95 and said VL of said first antigen binding site comprises said amino acid sequence of SEQ ID NO:85 or the protein of claim 1.
(Item 47)
47. The protein of any one of items 1-46, wherein said antibody Fc domain is a human IgG1 antibody Fc domain.
(Item 48)
48. The protein of item 47, wherein said antibody Fc domain or part thereof comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:118.
(Item 49)
Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, wherein at least one polypeptide chain of said antibody Fc domain is numbered according to the EU numbering system; Item 47 or 48 comprising one or more mutations compared to SEQ ID NO: 118 at one or more positions selected from K392, T394, D399, S400, D401, F405, Y407, K409, T411 and K439 Proteins as described in.
(Item 50)
at least one polypeptide chain of said antibody Fc domain Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q numbered according to the EU numbering system 、Ｅ３５７Ｌ、Ｅ３５７Ｗ、Ｋ３６０Ｅ、Ｋ３６０Ｗ、Ｑ３６２Ｅ、Ｓ３６４Ｋ、Ｓ３６４Ｅ、Ｓ３６４Ｈ、Ｓ３６４Ｄ、Ｔ３６６Ｖ、Ｔ３６６Ｉ、Ｔ３６６Ｌ、Ｔ３６６Ｍ、Ｔ３６６Ｋ、Ｔ３６６Ｗ、Ｔ３６６Ｓ、Ｌ３６８Ｅ、Ｌ３６８Ａ、Ｌ３６８Ｄ、Ｋ３７０Ｓ、Ｎ３９０Ｄ、Ｎ３９０Ｅ、Ｋ３９２Ｌ、Ｋ３９２Ｍ、Ｋ３９２Ｖ , K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K4139E, and 50. The protein of any one of items 47-49, comprising one or more mutations relative to SEQ ID NO:118.
(Item 51)
one polypeptide chain of said antibody heavy chain constant region is: comprises one or more mutations relative to SEQ ID NO: 118 at one or more positions selected from Y407, K409, T411, and K439; and said other polypeptide chain of said antibody heavy chain constant region is Q347, Y349, L351, S354, E356, E357, S364, T366, L368, K370, N390, K392, T394, D399, D401, F405, Y407, K409, T411 and K439 numbered according to the EU numbering system 51. The protein of any one of items 47-50, comprising one or more mutations compared to SEQ ID NO: 118 at one or more positions selected from.
(Item 52)
one polypeptide chain of said antibody heavy chain constant region comprises K360E and K409W substitutions relative to SEQ ID NO: 118; and said other polypeptide chain of said antibody heavy chain constant region is numbered according to the EU numbering system 52. The protein of item 51, comprising the Q347R, D399V and F405T substitutions compared to SEQ ID NO: 118, labeled with .
(Item 53)
one polypeptide chain of said antibody heavy chain constant region comprises a Y349C substitution relative to SEQ ID NO: 118; said other polypeptide chain of said antibody heavy chain constant region is numbered according to the EU numbering system; 53. The protein of item 51 or 52, which also comprises the S354C substitution as compared to SEQ ID NO:118.
(Item 54)
is a protein and:
(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:259 or SEQ ID NO:276;
(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:260; and
(c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:261;
Said protein.
(Item 55)
A pharmaceutical composition comprising the protein of any one of items 1-54 and a pharmaceutically acceptable carrier.
(Item 56)
A pharmaceutical formulation comprising the protein according to any one of items 1 to 54 and a buffer solution having a pH of 7.0 or higher.
(Item 57)
57. Pharmaceutical formulation according to item 56, wherein the pH of said formulation is between 7.0 and 8.0.
(Item 58)
58. Pharmaceutical formulation according to item 56 or 57, wherein said buffer comprises citrate.
(Item 59)
59. Pharmaceutical formulation according to item 58, wherein the concentration of said citrate is 10-20 mM.
(Item 60)
60. The pharmaceutical formulation of any one of items 56-59, wherein said buffer further comprises phosphate.
(Item 61)
60. Pharmaceutical formulation according to any one of items 56-59, further comprising a sugar or sugar alcohol.
(Item 62)
62. Pharmaceutical formulation according to item 61, wherein said sugar or sugar alcohol is a disaccharide.
(Item 63)
63. Pharmaceutical formulation according to item 62, wherein said disaccharide is sucrose.
(Item 64)
64. Pharmaceutical formulation according to any one of items 61-63, wherein said concentration of said sugar or sugar alcohol in said pharmaceutical formulation is 200-300 mM.
(Item 65)
65. The pharmaceutical formulation of item 64, wherein said concentration of said sugar or sugar alcohol in said pharmaceutical formulation is about 250 mM.
(Item 66)
Pharmaceutical formulation according to any one of items 56-65, further comprising a non-ionic surfactant.
(Item 67)
67. Pharmaceutical formulation according to item 66, wherein said non-ionic surfactant comprises polysorbate.
(Item 68)
68. Pharmaceutical formulation according to item 67, wherein said polysorbate is polysorbate 80.
(Item 69)
69. Pharmaceutical formulation according to item 68, wherein said concentration of polysorbate 80 in said pharmaceutical formulation is between 0.005% and 0.05%.
(Item 70)
70. The pharmaceutical formulation of item 69, wherein said concentration of polysorbate 80 in said pharmaceutical formulation is about 0.01%.
(Item 71)
71. The pharmaceutical formulation of any of items 56-70, wherein the concentration of NaCl, if present, is about 1 mM or less in said pharmaceutical formulation.
(Item 72)
72. Pharmaceutical formulation according to any one of items 56-71, wherein the concentration of said protein is 10-200 mg/mL.
(Item 73)
73. The pharmaceutical formulation of item 72, wherein the concentration of said protein is about 20 mg/mL.
(Item 74)
The pharmaceutical formulation of any one of items 56-59 and 61-73, comprising about 20 mM citrate, about 250 mM sucrose, and about 0.01% polysorbate 80 at pH 7.0.
(Item 75)
74. The method of any one of items 56-60 and 72-73, comprising about 20 mg/mL protein, about 20 mM potassium phosphate, about 10 mM sodium citrate, and about 125 mM sodium chloride at pH 7.8. Pharmaceutical formulation.
(Item 76)
A cell comprising one or more nucleic acids encoding a protein according to any one of items 1-54.
(Item 77)
A method of enhancing tumor cell death, wherein tumor cells and natural killer cells are treated with an effective amount of the protein of any one of items 1-54, the pharmaceutical composition of item 55, or the pharmaceutical composition of items 56-75. A method comprising exposure to a pharmaceutical formulation according to any one of
(Item 78)
A method of treating cancer comprising administering to a subject in need thereof an effective amount of the protein of any one of items 1-54, the pharmaceutical composition of item 55, or any of items 56-75 The method, comprising administering the pharmaceutical formulation of paragraph 1.
(Item 79)
79. The method of item 78, wherein the cancer is a hematological malignancy.
(Item 80)
The hematologic malignancies include acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), myeloproliferative neoplasms (MPN), lymphoma, non-Hodgkin's lymphoma, and classical 80. The method of item 79, wherein the method is selected from the group consisting of:
(Item 81)
The AML is undifferentiated acute myeloblastic leukemia, minimally differentiated acute myeloblastic leukemia, differentiated acute myeloblastic leukemia, acute promyelocytic leukemia (APL), acute myelomonocytic leukemia, acute myelomonocytic leukemia with eosinophilia, acute monocytic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia (AMKL), acute basophilic leukemia, acute panmyelopathy with fibrosis, and blast plasmacytoid dendritic cell neoplasm (BPDCN).
(Item 82)
82. The method of any one of items 80 or 81, wherein said AML is characterized by expression of CLL-1 on AML leukemia stem cells (LSC).
(Item 83)
83. The method of item 82, wherein said LSCs further express cell membrane markers selected from CD34, CD38, CD123, TIM3, CD25, CD32, and CD96.
(Item 84)
84. The method of any one of items 80-83, wherein said AML is minimal residual disease (MRD).
(Item 85)
The MRD comprises FLT3-ITD ((Fms-like tyrosine kinase 3)-intragenic tandem duplication (ITD)), NPM1 (nucleophosmin 1), DNMT3A (DNA methyltransferase gene DNMT3A), and IDH (isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2)).
(Item 86)
The MDS is MDS with multilineage dysplasia (MDS-MLD), MDS with single lineage dysplasia (MDS-SLD), MDS with ringed sideroblasts (MDS-RS), increased blasts 81. The method of item 80, wherein the method is selected from MDS with isolated del (MDS-EB), MDS with isolated del (5q), and unclassified MDS (MDS-U).
(Item 87)
81. The method of item 80, wherein the MDS is primary MDS or secondary MDS.
(Item 88)
81. The method of item 80, wherein said ALL is selected from B-cell acute lymphoblastic leukemia (B-ALL) and T-cell acute lymphoblastic leukemia (T-ALL).
(Item 89)
81. The method of item 80, wherein said MPN is selected from polycythemia vera, essential thrombocythemia (ET), and myelofibrosis.
(Item 90)
81. The method of item 80, wherein said non-Hodgkin's lymphoma is selected from B-cell lymphoma and T-cell lymphoma.
(Item 91)
The lymphoma includes chronic lymphocytic leukemia (CLL), lymphoblastic lymphoma (LPL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), primary mediastinal large B-cell lymphoma ( PMBL), follicular lymphoma, mantle cell lymphoma, pilocytic leukemia, plasma cell myeloma (PCM) or multiple myeloma (MM), mature T/NK cell tumors, and histiocytic tumors 80. The method according to 80.
(Item 92)
92. The method of any one of items 78-91, wherein said cancer expresses CLEC12A.

Claims (92)

is a protein and:
(a) a first antigen binding site that binds to NKG2D;
(b) a second antigen binding site that binds CLEC12A; and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen binding site that binds CD16;
wherein said second antigen-binding site that binds to CLEC12A is:
(i) a heavy chain comprising complementarity determining region 1 (CDR1), complementarity determining region 2 (CDR2), and complementarity determining region 3 (CDR3), each comprising the amino acid sequences of SEQ ID NOS: 137, 272, and 273; a variable domain (VH); and a light chain variable domain (VL) comprising CDR1, CDR2 and CDR3, each comprising the amino acid sequences of SEQ ID NOs: 140, 141 and 142;
(ii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 137, 138 and 139 respectively; VL containing;
(iii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOS: 251, 196 and 246 respectively; VL containing;
(iv) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:221, 166 and 223 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:240, 226 and 179 respectively VL containing;
(v) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:206, 166 and 241 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:245, 239 and 179 respectively VL containing;
(vi) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:221, 236 and 223 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:152, 226 and 179 respectively; VL containing;
(vii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 159, 170 and 183 respectively; VL containing;
(viii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 197, 202 and 207 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 211, 212 and 214 respectively VL;
(ix) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:220, 222 and 257 respectively; and a CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs:224, 225 and 227 respectively; or (x) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOS:230, 231 and 232 respectively; and CDR1 comprising the amino acid sequences of SEQ ID NOS:233, 234 and 235 respectively , CDR2, and VL containing CDR3. 2. The protein of claim 1, wherein said second antigen binding site that binds CLEC12A comprises
(i) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 272, and 273, respectively; or (ii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOS: 137, 138 and 139 respectively; and VL containing CDR3,
The protein, comprising: 3. A protein according to claim 1 or 2, wherein
(i) said VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:335, and said VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:336;
(ii) said VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:270, and said VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:271; or (iii) said VH comprises , said protein comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:178, and said VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:289. A protein according to any one of claims 1 to 3,
(i) said VH comprises the amino acid sequence of SEQ ID NO:335 and said VL comprises the amino acid sequence of SEQ ID NO:336;
(ii) said VH comprises the amino acid sequence of SEQ ID NO:270 and said VL comprises the amino acid sequence of SEQ ID NO:271;
(iii) said protein wherein said VH comprises the amino acid sequence of SEQ ID NO:178 and said VL comprises the amino acid sequence of SEQ ID NO:289; 143 and 144; 178 and 144; 256 and 144; 143 and 163; 143 and 167; 143 and 171; , a protein according to claim 1 or 2. Said second antigen binding site is present as a single chain fragment variable (scFv), wherein said scFv comprises 6. The protein of any one of claims 1-5, comprising an amino acid sequence selected from 161, 164, 165, 168, 169, 172, 173, 176, 177, 180, and 181. 7. The protein of claim 6, wherein said scFv comprises the amino acid sequence of SEQ ID NO:274 or claim 180. a VH wherein said second antigen-binding site that binds CLEC12A comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs: 251, 196, and 246, respectively; and amino acids of SEQ ID NOs: 198, 199, and 201, respectively. 2. The protein of claim 1, comprising a VL comprising CDRl, CDR2, and CDR3 comprising sequences. wherein said second antigen binding site that binds to CLEC12A is:
(i) VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 195, 196, 187 respectively; VL comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 198, 199, 201 respectively; ;
(ii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 192, 196 and 187 respectively; or (iii) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOs: 192, 196 and 213 respectively; and CDR1 comprising the amino acid sequences of SEQ ID NOs: 198, 199 and 201 respectively. VL containing CDR2 and CDR3,
2. The protein of claim 1, comprising: Any one of claims 1 and 8-9, wherein said VH comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 148 and said VL comprises an amino acid sequence that is at least 90% identical to SEQ ID NO: 203 The protein according to the paragraph. The protein of any one of claims 1 and 8-10, wherein said VH comprises the amino acid sequence of SEQ ID NO:148 and said VL comprises the amino acid sequence of SEQ ID NO:203. 210 and 203; 162 and 218; 148 and 218; or 210 and 218. said second antigen binding site is present as a scFv, wherein said scFv is selected from SEQ ID NOs: 184, 185, 204, 205, 208, 209, 215, 216, 252, 253, 254, and 255 13. The protein of any one of claims 8-12, comprising the amino acid sequence of 14. The protein of claim 13, wherein said scFv comprises the amino acid sequence of SEQ ID NO:204. 4. The protein of any one of the preceding claims, wherein said second antigen binding site binds CLEC12A in a glycosylation-independent manner. is a protein and:
(a) a first antigen binding site that binds NKG2D;
(b) a second antigen binding site that binds CLEC12A; and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen binding site that binds CD16,
wherein said second antigen binding site binds to CLEC12A in a glycosylation-independent manner. 17. The protein of any one of claims 1-16, wherein said second antigen binding site binds human CLEC12A with a dissociation constant (K _D ) of 20 nM or less, as measured by surface plasmon resonance (SPR). 18. The protein of any one of claims 1-7 and 16-17, wherein said second antigen binding site binds human CLEC12A with a K _D of 1 nM or less as measured by SPR. The protein of any one of claims 1-7 and 15-18, wherein said second antigen binding site binds human CLEC12A comprising a K244Q substitution. A protein according to any preceding claim, wherein said protein comprises an antibody Fc domain, or a portion thereof sufficient to bind CD16. 21. The protein of any one of claims 1-20, wherein said first antigen binding site that binds NKG2D is a Fab fragment and said second antigen binding site that binds CLEC12A is a scFv. any one of claims 1-5, 8-12 and 15-20, wherein said first antigen binding site that binds NKG2D is a scFv and said second antigen binding site that binds CLEC12A is a Fab fragment The protein according to the paragraph. 21. The protein of any one of claims 1-20, further comprising an additional antigen binding site that binds CLEC12A. 24. The protein of claim 23, wherein said first antigen binding site that binds NKG2D is a scFv and said second and said additional antigen binding site that binds CLEC12A are each Fab fragments. 24. The protein of claim 23, wherein said first antigen binding site that binds NKG2D is a scFv and said second and said additional antigen binding site that binds CLEC12A are each a scFv. 26. The protein of any one of claims 23-25, wherein the amino acid sequences of said second and said additional antigen binding sites are identical. is a protein and:
(a) a first antigen binding site that binds to NKG2D;
(b) a second antigen binding site that binds CLEC12A; and (c) an antibody Fc domain or portion thereof sufficient to bind CD16, or a third antigen binding site that binds CD16,
wherein said first antigen binding site that binds NKG2D is a Fab fragment and said second antigen binding site that binds CLEC12A is a scFv. 22, 24-26, wherein said scFv that binds NKG2D is linked via a hinge containing Ala-Ser or Gly-Ser to an antibody constant domain or part thereof sufficient to bind CD16 The protein according to any one of Claims. Claims 21 and 25, wherein each scFv that binds CLEC12A is linked via a hinge containing an Ala-Ser or Gly-Ser to an antibody constant domain or portion thereof sufficient to bind CD16. 29. The protein of any one of claims 1-28. 30. The protein of claim 28 or 29, wherein said hinge further comprises the amino acid sequence Thr-Lys-Gly. any one of claims 22, 24-26, 28, and 30, wherein within said scFv that binds to NKG2D, said scFv heavy chain variable domain forms a disulfide bridge with said scFv light chain variable domain the indicated protein. any of claims 21, 25-27, and 30-31, wherein within each scFv that binds CLEC12A, said heavy chain variable domain of said scFv forms a disulfide bridge with said light chain variable domain of said scFv 1. Protein according to item 1. 33. The protein of claim 31 or 32, wherein said disulfide bridge is formed between C44 of said heavy chain variable domain and C100 of said light chain variable domain, numbered under the Kabat numbering scheme. any one of claims 22, 24-26, 28, 30-31 and 33, wherein within said scFv that binds NKG2D said heavy chain variable domain is linked to said light chain variable domain via a flexible linker The protein according to the paragraph. any of claims 21, 25-27, 29-30, and 32-34, wherein within each scFv that binds CLEC12A, said heavy chain variable domain is linked to said light chain variable domain via a flexible linker or the protein of claim 1. 36. The protein of claim 34 or 35, wherein said flexible linker comprises ( _G4S ) ₄ . any of claims 22, 24-26, 28, 30-31, 33-34, and 36, wherein within said scFv that binds NKG2D said heavy chain variable domain is located C-terminal to said light chain variable domain 1. Protein according to item 1. any of claims 21, 25-27, 29-30, 32-33, and 35-37, wherein within each scFv that binds CLEC12A, said heavy chain variable domain is located C-terminal to said light chain variable domain or the protein of claim 1. any of claims 22, 24-26, 28, 30-31, 33-34, and 36, wherein within said scFv that binds NKG2D said heavy chain variable domain is located N-terminal to said light chain variable domain 1. Protein according to item 1. claims 21, 25-27, 29-30, 32-33, 35-36 and 39, wherein within each scFv that binds CLEC12A said heavy chain variable domain is located N-terminal to said light chain variable domain The protein according to any one of Claims. any of claims 21, 27, 29-30, 32-33, 35-36, 38 and 40, wherein the Fab fragment that binds NKG2D is not positioned between the antigen binding site and said Fc or portion thereof 1. Protein according to item 1. claims 22, 24, 26, 28, 30-31, 33-34, 36-37, and 39, wherein the Fab fragment that binds CLEC12A is not positioned between the antigen binding site and said Fc or portion thereof The protein according to any one of Claims. a VH wherein said first antigen-binding site that binds NKG2D comprises CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:81, 82, and 112, respectively; and amino acids of SEQ ID NOs:86, 77, and 87, respectively. 43. The protein of any one of claims 1-42, comprising a VL comprising CDR1, CDR2, and CDR3 comprising sequences. The first antigen-binding site that binds to NKG2D is
(i) a VH comprising CDR1, CDR2 and CDR3 comprising the amino acid sequences of SEQ ID NOS:81, 82 and 97, respectively; or (ii) a VH comprising CDR1, CDR2, and CDR3 comprising the amino acid sequences of SEQ ID NOs:81, 82, and 84, respectively; and CDR1, comprising the amino acid sequences of SEQ ID NOs:86, 77, and 87, respectively. , CDR2, and VL comprising CDR3;
The protein of any one of claims 1-42, comprising said VH of said first antigen binding site comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:95, and said VL of said first antigen binding site is an amino acid that is at least 90% identical to SEQ ID NO:85 45. A protein according to any of claims 43 or 44, comprising a sequence. of claims 43-45, wherein said VH of said first antigen binding site comprises said amino acid sequence of SEQ ID NO:95 and said VL of said first antigen binding site comprises said amino acid sequence of SEQ ID NO:85 A protein according to any one of claims 1 to 3. 47. The protein of any one of claims 1-46, wherein said antibody Fc domain is a human IgG1 antibody Fc domain. 48. The protein of claim 47, wherein said antibody Fc domain or portion thereof comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:118. Q347, Y349, L351, S354, E356, E357, K360, Q362, S364, T366, L368, K370, N390, wherein at least one polypeptide chain of said antibody Fc domain is numbered according to the EU numbering system; 47 or 48. The protein according to 48. at least one polypeptide chain of said antibody Fc domain Q347E, Q347R, Y349S, Y349K, Y349T, Y349D, Y349E, Y349C, L351K, L351D, L351Y, S354C, E356K, E357Q numbered according to the EU numbering system 、Ｅ３５７Ｌ、Ｅ３５７Ｗ、Ｋ３６０Ｅ、Ｋ３６０Ｗ、Ｑ３６２Ｅ、Ｓ３６４Ｋ、Ｓ３６４Ｅ、Ｓ３６４Ｈ、Ｓ３６４Ｄ、Ｔ３６６Ｖ、Ｔ３６６Ｉ、Ｔ３６６Ｌ、Ｔ３６６Ｍ、Ｔ３６６Ｋ、Ｔ３６６Ｗ、Ｔ３６６Ｓ、Ｌ３６８Ｅ、Ｌ３６８Ａ、Ｌ３６８Ｄ、Ｋ３７０Ｓ、Ｎ３９０Ｄ、Ｎ３９０Ｅ、Ｋ３９２Ｌ、Ｋ３９２Ｍ、Ｋ３９２Ｖ , K392F, K392D, K392E, T394F, D399R, D399K, D399V, S400K, S400R, D401K, F405A, F405T, Y407A, Y407I, Y407V, K409F, K409W, K409D, T411D, T411E, K4139E, and 50. The protein of any one of claims 47-49, comprising one or more mutations relative to SEQ ID NO:118. one polypeptide chain of said antibody heavy chain constant region is: comprises one or more mutations relative to SEQ ID NO: 118 at one or more positions selected from Y407, K409, T411, and K439; and said other polypeptide chain of said antibody heavy chain constant region is Q347, Y349, L351, S354, E356, E357, S364, T366, L368, K370, N390, K392, T394, D399, D401, F405, Y407, K409, T411 and K439 numbered according to the EU numbering system 51. The protein of any one of claims 47-50, comprising one or more mutations compared to SEQ ID NO: 118 at one or more positions selected from. one polypeptide chain of said antibody heavy chain constant region comprises K360E and K409W substitutions relative to SEQ ID NO: 118; and said other polypeptide chain of said antibody heavy chain constant region is numbered according to the EU numbering system 52. The protein of claim 51, comprising the Q347R, D399V and F405T substitutions compared to SEQ ID NO: 118 labeled with . one polypeptide chain of said antibody heavy chain constant region comprises a Y349C substitution relative to SEQ ID NO: 118; said other polypeptide chain of said antibody heavy chain constant region is numbered according to the EU numbering system; 53. The protein of claim 51 or 52, further comprising the S354C substitution as compared to SEQ ID NO:118. is a protein and:
(a) a first polypeptide comprising the amino acid sequence of SEQ ID NO:259 or SEQ ID NO:276;
(b) a second polypeptide comprising the amino acid sequence of SEQ ID NO:260; and (c) a third polypeptide comprising the amino acid sequence of SEQ ID NO:261.
Said protein. A pharmaceutical composition comprising the protein of any one of claims 1-54 and a pharmaceutically acceptable carrier. A pharmaceutical formulation comprising the protein according to any one of claims 1 to 54 and a buffer solution of pH 7.0 or higher. 57. Pharmaceutical formulation according to claim 56, wherein the pH of said formulation is between 7.0 and 8.0. 58. The pharmaceutical formulation of claim 56 or 57, wherein said buffer comprises citrate. 59. The pharmaceutical formulation of claim 58, wherein the citrate concentration is 10-20 mM. 60. The pharmaceutical formulation of any one of claims 56-59, wherein said buffer further comprises phosphate. 60. Pharmaceutical formulation according to any one of claims 56-59, further comprising a sugar or sugar alcohol. 62. The pharmaceutical formulation of claim 61, wherein said sugar or sugar alcohol is a disaccharide. 63. The pharmaceutical formulation of claim 62, wherein said disaccharide is sucrose. The pharmaceutical formulation of any one of claims 61-63, wherein said concentration of said sugar or sugar alcohol in said pharmaceutical formulation is 200-300 mM. 65. The pharmaceutical formulation of claim 64, wherein said concentration of said sugar or sugar alcohol in said pharmaceutical formulation is about 250 mM. Pharmaceutical formulation according to any one of claims 56-65, further comprising a non-ionic surfactant. 67. The pharmaceutical formulation of claim 66, wherein said non-ionic surfactant comprises polysorbate. 68. The pharmaceutical formulation of claim 67, wherein said polysorbate is polysorbate 80. 69. The pharmaceutical formulation of claim 68, wherein said concentration of polysorbate 80 in said pharmaceutical formulation is between 0.005% and 0.05%. 70. The pharmaceutical formulation of claim 69, wherein said concentration of polysorbate 80 in said pharmaceutical formulation is about 0.01%. 71. The pharmaceutical formulation of any of claims 56-70, wherein the concentration of NaCl, if present, is about 1 mM or less in said pharmaceutical formulation. A pharmaceutical formulation according to any one of claims 56 to 71, wherein the concentration of said protein is between 10 and 200 mg/mL. 73. The pharmaceutical formulation of claim 72, wherein the protein concentration is about 20 mg/mL. 74. The pharmaceutical formulation of any one of claims 56-59 and 61-73, comprising about 20 mM citrate, about 250 mM sucrose, and about 0.01% polysorbate 80 at pH 7.0. Claims 56-60 and 72-73, comprising about 20 mg/mL protein, about 20 mM potassium phosphate, about 10 mM sodium citrate, and about 125 mM sodium chloride at pH 7.8. pharmaceutical formulations of A cell comprising one or more nucleic acids encoding a protein according to any one of claims 1-54. A method of enhancing tumor cell death, comprising treating tumor cells and natural killer cells with an effective amount of the protein of any one of claims 1-54, the pharmaceutical composition of claim 55, or claim 55. A method comprising exposure to a pharmaceutical formulation according to any one of 56-75. A method of treating cancer, comprising administering to a subject in need thereof an effective amount of the protein of any one of claims 1-54, the pharmaceutical composition of claim 55, or claims 56-75 The method comprising administering the pharmaceutical formulation of any one of 79. The method of claim 78, wherein said cancer is a hematological malignancy. The hematologic malignancies include acute myelogenous leukemia (AML), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), myeloproliferative neoplasms (MPN), lymphoma, non-Hodgkin's lymphoma, and classical 80. The method of claim 79, wherein the method is selected from the group consisting of aggressive Hodgkin's lymphoma. The AML is undifferentiated acute myeloblastic leukemia, minimally differentiated acute myeloblastic leukemia, differentiated acute myeloblastic leukemia, acute promyelocytic leukemia (APL), acute myelomonocytic leukemia, acute myelomonocytic leukemia with eosinophilia, acute monocytic leukemia, acute erythroleukemia, acute megakaryoblastic leukemia (AMKL), acute basophilic leukemia, acute panmyelopathy with fibrosis, and blast plasmacytoid dendritic cell neoplasm (BPDCN). 82. The method of any one of claims 80 or 81, wherein said AML is characterized by expression of CLL-1 on AML leukemia stem cells (LSC). 83. The method of claim 82, wherein said LSCs further express cell membrane markers selected from CD34, CD38, CD123, TIM3, CD25, CD32, and CD96. 84. The method of any one of claims 80-83, wherein said AML is minimal residual disease (MRD). The MRD comprises FLT3-ITD ((Fms-like tyrosine kinase 3)-intragenic tandem duplication (ITD)), NPM1 (nucleophosmin 1), DNMT3A (DNA methyltransferase gene DNMT3A), and IDH (isocitrate dehydrogenase 1 and 85. The method of claim 84, characterized by the presence or absence of a mutation selected from 2 (IDH1 and IDH2). The MDS is MDS with multilineage dysplasia (MDS-MLD), MDS with single lineage dysplasia (MDS-SLD), MDS with ringed sideroblasts (MDS-RS), increased blasts 81. The method of claim 80 selected from MDS with MDS (MDS-EB), MDS with isolated del (5q), and unclassified MDS (MDS-U). 81. The method of claim 80, wherein the MDS is primary MDS or secondary MDS. 81. The method of claim 80, wherein said ALL is selected from B-cell acute lymphoblastic leukemia (B-ALL) and T-cell acute lymphoblastic leukemia (T-ALL). 81. The method of claim 80, wherein said MPN is selected from polycythemia vera, essential thrombocythemia (ET), and myelofibrosis. 81. The method of claim 80, wherein said non-Hodgkin's lymphoma is selected from B-cell lymphoma and T-cell lymphoma. The lymphoma includes chronic lymphocytic leukemia (CLL), lymphoblastic lymphoma (LPL), diffuse large B-cell lymphoma (DLBCL), Burkitt's lymphoma (BL), primary mediastinal large B-cell lymphoma ( PMBL), follicular lymphoma, mantle cell lymphoma, pilocytic leukemia, plasma cell myeloma (PCM) or multiple myeloma (MM), mature T/NK cell tumors, and histiocytic tumors. 81. The method of Paragraph 80. 92. The method of any one of claims 78-91, wherein said cancer expresses CLEC12A.

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